Diagnosis methods, diagnostic agents, and therapeutic agents against alzheimer&#39;s disease and frontotemporal lobar degeneration, and screening methods for these agents

ABSTRACT

It has been revealed that, from a pre-onset stage of Alzheimer&#39;s disease, enhancement of phosphorylations of MARCKS and the like causes abnormal spine formation or the like, consequently developing the disease. It also has been revealed that the phosphorylations of MARCKS and the like are caused by PKC and the like, and further that b-raf is involved in phosphorylation of tau protein important for the progression of Alzheimer&#39;s disease. Thus, these proteins have been found to be target molecules useful in the diagnosis and treatment of Alzheimer&#39;s disease. In addition, it has also been revealed that, in a pre-onset stage of frontotemporal lobar degeneration, b-RAF phosphorylation enhancement causes a decrease in the number of spines and the like, consequently developing the disease. Thus, b-RAF has been found to be a target molecule useful in the diagnosis and treatment of frontotemporal lobar degeneration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/107,502, filed on Sep. 23, 2016, which is National Stage ofInternational Application No. PCT/JP2014/084424, filed on Dec. 25, 2014,which claims priority from Japanese Patent Application No. 2013-272189,filed on Dec. 27, 2013, the contents of all of which are incorporatedherein by reference their entirety.

TECHNICAL FIELD

The present invention relates to a diagnosis method, a diagnostic agent,and a therapeutic agent against Alzheimer's disease. Further, thepresent invention relates to a screening method for candidate compoundsof these agents. Moreover, the present invention relates to a diagnosticagent and a therapeutic agent against frontotemporal lobar degeneration.

BACKGROUND ART

Alzheimer's disease (Alzheimer's dementia, AD) is a progressiveneurodegenerative disease that may occur in presenile to senile stages.The main symptoms include memory disorder, higher brain functiondisorders (aphasia, apraxia, agnosia, constructional apraxia), change inpersonality, and so forth. In addition, because of such symptoms, thedisease not only reduces the quality of life of a patienthimself/herself, but also greatly influences the living styles of familyand so on around the patient. Further, the number of the patients issteadily increasing along with population ageing. Alzheimer's disease isa serious problem of modern society all over the world. Hence,Alzheimer's disease has been studied actively, but the elucidation ofthe full onset mechanism thereof and the development of an eradicativemedicine have not been achieved yet under current situations.

On the other hand, it is now possible to delay the progression ofAlzheimer's disease symptoms more than ever. Particularly,cholinesterase inhibitors have been actually used in clinical settings,resulting in some reasonable outcomes. The progression of the symptomscan also be suppressed to some degree currently. As a result, there is ademand in the treatment of Alzheimer's disease that the disease shouldbe detected at earlier stages, thereby hastening developments of:electroencephalography, biochemical tests targeting blood andcerebrospinal fluid, diagnostic imagings such as CT, MRI, and PET/SPECT,and so forth. Particularly, PET is about to enable the detection ofsenile plaque (amyloid plaque) deposition in the brain, which is acharacteristic of Alzheimer's disease and the most likely causativefactor thereof. However, currently-available diagnostic techniques stillhave difficulty grasping a pre-onset stage of Alzheimer's disease isdeveloped, and no effective early-stage diagnosis method has beenestablished yet under current situations.

Besides senile plaque deposition, Alzheimer's disease isneuropathologically characterized also by neurofibrillary tangle (pairedhelical filament (PHF)) deposition. In addition, the deposition of thesestructures is believed to cause nerve function disorder and nerve celldeath (nerve cell dropout) involved in the symptoms described above.Moreover, it has been revealed that senile plaques are structures formedwhen polypeptides, each composed of approximately 40 amino acids, calledamyloid β (Aβ) aggregate and deposit outside nerve cells in highdensity. Further, neurofibrillary tangles have been revealed to be alsostructures formed when microtubule-associated proteins tau arephosphorylated and thereby dissociated from cytoskeleton-formingmicrotubules, followed by polymerization among the tau proteins.Meanwhile, although no conclusion has been drawn yet regarding theAlzheimer causative factor and onset mechanism, the most likelymechanism is such that when amyloid β molecules aggregate (amyloidpathology), the aggregation promotes the tau phosphorylation andpolymerization (tau pathology), consequently leading to nerve cell deathand so forth (amyloid cascade hypothesis).

Furthermore, it is suggested that various phosphorylation signaltransductions are involved in a pathology of Alzheimer's disease. Forexample, as described above, the deposition of neurofibrillary tanglesis due to tau phosphorylation. It is also revealed that this tauphosphorylation is regulated by various serine/threonine kinases such asGSK3β, JNK, PKA, Cdk5, and casein kinase II (NPLs 1 to 7). Moreover, ithas been presumed that microtubules from which tau is dissociated bysuch phosphorylation become unstable, consequently decreasing neuritesas observed in the brains of AD patients (NPLs 8 and 9).

In addition, it is suggested that a phosphorylation enzyme PKC isinvolved in memory formation (NPLs 10 and 11). Further, activating PKCand CaMKII is believed to promote the transcriptions of BDNF and Arcinvolved in memory control, and also have a protective function againstAlzheimer's disease (NPLs 12 and 13). Additionally, based on suchfindings, an attempt has been made to apply PKC activators such asbryostatin in the treatment of Alzheimer's disease. However, it is alsoreported that an excessive activation of PKC, on the other hand, impairsworking memory (NPL 14).

Furthermore, it is also suggested that the phosphorylation of MARCKS byPKC dissociates this protein from the cell membrane (PIP2 and actins)and, as a result, induces amyloid β production (NPL 15). Moreover,phosphorylated MARCKS is observed in dystrophic neurites and microgliawithin senile plaques. Nevertheless, it has also been revealed that thephosphorylation level of MARCKS in the brains of Alzheimer's diseasepatients is lower than that of healthy subjects (NPL 16).

As described above, it has been suggested that phosphorylation signaltransductions are involved in a pathology of Alzheimer's disease. Ifthis involvement can be elucidated in more details, it is expected togreatly contribute to establishments of early-stage diagnosis andtreatment methods against this disease.

Nonetheless, in a phosphorylation signal transduction, particularly, ina wide variety of phosphorylation signal transductions in Alzheimer'sdisease, what protein phosphorylations play a central role in apre-onset stage of Alzheimer's disease has not been elucidated at allyet.

Meanwhile, frontotemporal lobar degeneration (FTLD) is known as adisease that exhibits progressive neurodegenerative disorders likeAlzheimer's disease. Frontotemporal lobar degeneration is the second orthird most frequent early-onset neurodegenerative dementia afterAlzheimer's disease. The symptoms to be exhibited include drasticchanges in behavior and personality. A language function disorder occurstogether with FTLD in many cases, and gradually develops into acognitive disorder and dementia. In addition, the studies have beenconducted as in the case of Alzheimer's disease, but the full onsetmechanism of FTLD has not been revealed yet.

For example, it is known that one cause of genetic frontotemporal lobardegeneration is a mutation in the PGRN gene. Moreover, there is a reportthat the PGRN protein exhibits an antagonistic action against TNF inbinding to TNF receptors, suggesting that this antagonism is involved inthe onset of frontotemporal lobar degeneration (NPL 17). However, on theother hand, contradictory results are also reported. The molecularmechanism in the onset of frontotemporal lobar degeneration, includingthe possibility of the involvement of the TNF signal transductionpathway (NPLs 18 to 21), has not been elucidated under currentsituations.

Additionally, for the elucidation of the molecular mechanism offrontotemporal lobar degeneration, PGRN gene knockout mice have beenprepared as model animals. Moreover, findings (such as excessiveinflammatory reaction, cellular ageing, synaptic dysfunction,ubiquitination promotion, increased caspase activation, decreased TDP-43solubility) exhibited in frontotemporal lobar degeneration are actuallyobserved in such knockout mice (NPLs 22 to 28). However, as has beenpointed out in other neurodegenerative diseases such as Alzheimer'sdisease also (NPL 29), artificially reducing the amount of the PGRNprotein in the model animals actually results in mere mimicking ofsymptoms caused by the expressions of mutated PGRN mRNA and the like, sothat the effectiveness as model animals is questionable.

As has been described above, no effective model animal is developedagainst frontotemporal lobar degeneration, and the onset mechanism hasnot been elucidated as in the case of Alzheimer's disease. Hence, in thedevelopment of diagnosis and treatment methods against the disease, nouseful target molecule has been found under current situations.

CITATION LIST [Non Patent Literatures]

-   [NPL 1] Masliah, E. et al., Am. J. Pathol., 1992, Vol. 140, pp. 263    to 268-   [NPL 2] Shoji, M. et al., Brain Res. Mol. Brain Res., 2000, Vol. 85,    pp. 221 to 233-   [NPL 3] Zhu, X. et al., J. Neurochem., 2003, Vol. 85, pp. 87 to 93-   [NPL 4] Takashima, A. et al., Proc. Natl. Acad. Sci. U.S.A., 1993,    Vol. 90, pp. 7789 to 7793-   [NPL 5] Hanger, D. P. et al., J. Biol. Chem., 2007, Vol. 282, pp.    23645 to 23654-   [NPL 6] Wang, J. Z. et al., Eur. J. Neurosci., 2007, Vol. 25, pp. 59    to 68-   [NPL 7] Piedrahita, D. et al., J. Neurosci., 2010, Vol. 30, pp.    13966 to 13976-   [NPL 8] Alonso, A. C. et al., Proc. Natl. Acad. Sci. U.S.A., 1994,    Vol. 91, pp. 5562 to 5566-   [NPL 9] Iqbal, K. et al., Lancet, 1986, Vol. 2, pp. 421 to 426-   [NPL 10] Abeliovich, A. et al., Cell, 1993, Vol. 75, pp. 1253 to    1262-   [NPL 11] Abeliovich, A. et al., Cell, 1993, Vol. 75, pp. 1263 to    1271-   [NPL 12] Orsini, C. A. et al., Neurosci. Biobehav. Rev., Vol. 36,    2012, pp. 1773 to 1802-   [NPL 13] Alkon, D. L. et al., Trends Pharmacol. Sci., 2007, Vol. 28,    pp. 51 to 60-   [NPL 14] Birnbaum, S. G. et al., Science, 2004, Vol. 29, pp. 882 to    884-   [NPL 15] Rui S U et al., Neurosci Bull, 2010, Vol. 26, pp. 338 to    344-   [NPL 16] Kimura T. et al., Neuroreport., 2000, Vol. 11, pp. 869 to    73-   [NPL 17] Tang, W. et al., Science, 2011, Vol. 332, pp. 478 to 484-   [NPL 18] Chen, X. et al., J. Neurosci., 2013, Vol. 33, pp. 9202 to    9213,-   [NPL 19] Jian, J. et al., FEBS Lett., 2013, Vol. 587, pp. 3428 to    3436,-   [NPL 20] Etemadi, N. et al., Immunol. Cell Biol., 2013, Vol. 91, pp.    661 to 664-   [NPL 21] Hu, Y. et al., Immunology, 2014, Vol. 142, pp. 193 to 201-   [NPL 22] Martens, L. H. et al., J. Clin. Invest., 2012, Vol. 122,    pp. 3955 to 3959-   [NPL 23] Yin, F. et al., FASEB J., 2010, Vol. 24, pp. 4639 to 4647-   [NPL 24] Yin, F. et al., J. Exp. Med., 2010, Vol. 207, pp. 117 to    128-   [NPL 25] Wils, H. et al., J. Pathol., 2012, Vol. 228, pp. 67 to 76-   [NPL 26] Petkau, T. L. et al., Neurobiol. Dis., 2012, Vol. 45, pp.    711 to 722-   [NPL 27] Ahmed, Z. et al., Am. J. Pathol., 2010, Vol. 177, pp. 311    to 324-   [NPL 28] Ghoshal, N. et al., Neurobiol. Dis., 2012, Vol. 45, pp. 395    to 408-   [NPL 29] Saito, T. et al., Nat. Neurosci., 2014, Vol. 17, pp. 661 to    663

SUMMARY Technical Problem

The present invention has been made in view of the problems of theabove-described conventional techniques. An object of the presentinvention is to identify phosphoproteins and kinase proteins which playcentral roles in a pre-onset stage of Alzheimer's disease, as well as anetwork composed of these proteins, and consequently to provide targetmolecules useful in the diagnosis and treatment of Alzheimer's disease.

In addition, another object of the present invention is to identify asignal transduction pathway which plays a central role in a pre-onsetstage of frontotemporal lobar degeneration, and consequently to providetarget molecules useful in the diagnosis and treatment of frontotemporallobar degeneration.

Solution to Problem

In order to provide target molecules useful in the diagnosis andtreatment of Alzheimer's disease, the present inventor first employed ananalysis according to a mass spectrometry method (2D LC MS/MS analysis)targeting brains at the pre-onset stage of tau model mice and four typesof Alzheimer's disease (AD) model mice and on postmortem brains of ADpatients, and searched 1100 or more phosphoproteins and 30000 or morephosphopolypeptides for proteins whose expression amounts changed incomparison with the respective wild-type mice and healthy subjects, forexample.

As a result, phosphoproteins whose expressions changed immediatelybefore or immediately after amyloid β started aggregating weresuccessfully identified in the brains of multiple AD model mice.Further, it was revealed that the phosphorylations of most of thesephosphoproteins also changed commonly in the AD patients or the taumodel mice. In sum, MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB weresuccessfully selected as proteins (AD core proteins) whosephosphorylation levels changed in brains affected with Alzheimer'sdisease, and which were presumed to play central roles in the pathology.

Further, these AD core proteins were incorporated into theexperimentally verified protein-protein interaction (PPI) database forthe analysis. Thus, an AD signaling network (AD core network) wasidentified which would serve as the core in the pathology of Alzheimer'sdisease.

The result surprisingly revealed that most of the AD core proteinsdirectly interacted with each other, and further that their functionswere focused on important functions in synapse such as spine formation,vesicle recycling, and energy metabolism. Particularly, it was revealedthat the enhancement of the phosphorylations of the AD core proteinsinvolved in nerve cell skeleton started from a non-symptomatic stagebefore amyloid β aggregation. Further, it was possible to categorize thechanges in the AD core protein phosphorylations in a pre-onset stage ofAlzheimer's disease into three patterns: one having a peak at an initialphase, one having a peak at a mid phase, and one having a peak at a latephase. In this manner, it was also revealed that the phosphorylationchanged in each AD core protein in a time-specific manner.

Further, the analysis utilizing the protein-protein interaction databasealso enables the identifications of PKC, CaMK, CSK, and Lyn as kinaseswhich controlled such time-specific changes in phosphorylations.

Meanwhile, it has been presumed that the transition from amyloid βaggregation (amyloid pathology) to tau phosphorylation andpolymerization (tau pathology) by the aggregation plays an importantrole in the pathology of Alzheimer's disease. In this regard, the resultof the mass spectrometry on the model mice also enabled theidentification of b-RAF as a kinase which promoted the transition fromamyloid pathology to tau pathology.

Furthermore, it was also verified that suppressing the expression ofMARCKS or suppressing the kinase activity of PKC or CaMK enabled arecovery of Alzheimer's disease pathology (abnormal spine formation).These have led to the completion of the present invention.

Additionally, in order to provide target molecules useful in thediagnosis and treatment of frontotemporal lobar degeneration (FTLD), thepresent inventor first made efforts to prepare an animal which could besaid as a true FTLD model in view of the current situation where theexpressions of mutated mRNA and the like, which would cause the disease,had not been reproduced by the existing FTLD model animals as describedabove. To be more specific, efforts were made to prepare FTLD model miceby introducing a stop mutation, which was observed in FTLD patients,into the PGRN (progranulin) gene of mice. As a result, it was found outnot only that the expressions of the mutated PGRN mRNA and a mutantprotein encoded thereby were observed in the obtained PGRN-KI mice, butalso that the introduction of the mutation enabled reproduction of boththe pathological observations and the clinical symptoms of FTLD patientsin the mice. Accordingly, it was revealed that the PGRN-KI mice werequite useful as FTLD model animals.

Next, using the PGRN-KI mice, efforts were made, as in the case of theabove Alzheimer's disease analysis, to comprehensively analyze(phosphoproteome analysis) phosphorylation signal transductions in FTLDalso to identify a phosphorylation signal transduction which played acentral role in a pathology of the disease.

As a result, surprisingly, it was found that no protein had a change inphosphorylation in a TNF signal transduction pathway per se which hadbeen heretofore suggested to be involved in the onset mechanism of FTLD.On the other hand, it was revealed that, in TNF-related signaltransduction pathways such as a MAPK signal transduction pathway in thePGRN-KI mice, the phosphorylations of proteins belonging to such signaltransduction pathways were remarkably changed. Particularly, a MAPKsignal transduction pathway was apparently activated in the PGRN-KI micefrom the pre-onset stage. During the period of symptom progression also,multiple proteins belonging to the signal transduction pathway were inhigh phosphorylation states all the time.

Hence, next, an analysis was performed for the therapeutic effect oftargeting b-RAF, its phosphorylation substrate tau, and the like, whichbelonged to the MAPK signal transduction pathway, and which wererevealed to be in high phosphorylation states in the PGRN-KI mice by theaforementioned analysis. To be more specific, first, analyzed waswhether or not suppressing an abnormal activation in the MAPK signaltransduction pathway by using a b-raf specific inhibitor or the likewould recover the phenotype of the PGRN-KI mice.

The result revealed that administering the b-raf inhibitor alleviatedthe abnormal behavior observed in the PGRN-KI mice. Further, it was alsorevealed that administering the b-raf inhibitor or knocking down taurecovered the number of spines which was decreased in the PGRN-KI mice.These have led to the completion of the present invention.

To be more specific, the present invention relates to a diagnosismethod, a diagnostic agent, a screening method for a candidate compoundof the diagnostic agent, a therapeutic agent, and a screening method fora candidate compound of the therapeutic agent all of which are againstAlzheimer's disease and target the above-described AD core proteins andkinases for phosphorylating the proteins. More specifically, the presentinvention provides the following.

<1> A method for diagnosing Alzheimer's disease, the method comprising:

(i) a step of detecting, in a test subject, a phosphorylation of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB;

(ii) a step of comparing the phosphorylation with a phosphorylation of asubstrate protein in a normal subject; and

(iii) a step of determining that the test subject is affected withAlzheimer's disease or has a risk of developing Alzheimer's disease ifthe phosphorylation of the substrate protein in the test subject ishigher than the phosphorylation of the substrate protein in the normalsubject as a result of the comparison.

<2> A method for diagnosing Alzheimer's disease, the method comprising:

(i) a step of detecting an activity or expression of a kinase protein ina test subject;

(ii) a step of comparing the activity or expression with an activity orexpression of a kinase protein in a normal subject; and

(iii) a step of determining that the test subject is affected withAlzheimer's disease or has a risk of developing Alzheimer's disease ifthe activity or expression of the kinase protein in the test subject ishigher than the activity or expression of the kinase protein in thenormal subject as a result of the comparison, wherein

the kinase protein is at least one kinase protein selected from thegroup consisting of PKC, CaMK, CSK, Lyn, and b-RAF.

<3> An agent for diagnosing Alzheimer's disease, the agent comprising acompound having an activity of binding to a phosphorylation site of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB.<4> An agent for diagnosing Alzheimer's disease, the agent comprising acompound having an activity of binding to at least one kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF.<5> A screening method for a candidate compound for diagnosingAlzheimer's disease, the method comprising the steps of:

bringing a test compound into contact with a phosphorylation site of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and

selecting the compound if the compound binds to the phosphorylationsite.

<6> A screening method for a candidate compound for diagnosingAlzheimer's disease, the method comprising the steps of:

bringing a test compound into contact with at least one kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF;and

selecting the compound if the compound binds to the kinase protein.

<7> An agent for treating Alzheimer's disease, the agent comprising acompound capable of suppressing a phosphorylation of at least onesubstrate protein selected from the group consisting of MARCKS,Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH,NFL, GPRIN1, ACON, ATPA, and ATPB.<8> An agent for treating Alzheimer's disease, the agent comprising acompound capable of suppressing an activity or expression of at leastone kinase protein selected from the group consisting of PKC, CaMK, CSK,Lyn, and b-RAF.<9> The agent according to <8>, wherein the compound is capable ofsuppressing an activity or expression of b-RAF and is at least onecompound selected from the group consisting of PLX-4720, sorafenib,GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate, and LGX818.<10> The agent according to <9>, wherein the compound is vemurafenib.<11> An agent for treating Alzheimer's disease, the agent comprising acompound capable of suppressing a binding of at least one substrateprotein selected from the group consisting of MARCKS, Marcksl1, SRRM2,SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON,ATPA, and ATPB to at least one kinase protein selected from the groupconsisting of PKC, CaMK, CSK, Lyn, and b-RAF.<12> A screening method for a candidate compound for treatingAlzheimer's disease, the method comprising:

(i) a step of applying a test compound to a system capable of detectinga phosphorylation of at least one substrate protein selected from thegroup consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and

(ii) a step of selecting the compound if the compound suppresses thephosphorylation of the substrate protein.

<13> A screening method for a candidate compound for treatingAlzheimer's disease, the method comprising:

(i) a step of applying a test compound to a system capable of detectingan activity or expression of at least one kinase protein selected fromthe group consisting of PKC, CaMK, CSK, Lyn, and b-RAF; and

(ii) a step of selecting the compound if the compound suppresses theactivity or expression of the protein.

<14> A screening method for a candidate compound for treatingAlzheimer's disease, the method comprising the following steps (a) to(c):

(a) a step of bringing at least one kinase protein selected from thegroup consisting of PKC, CaMK, CSK, Lyn, and b-RAF into contact with atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB, in presence of a testcompound;

(b) a step of detecting a binding between the kinase protein and thesubstrate protein; and

(c) a step of selecting the compound if the compound suppresses thebinding.

In addition, the present invention relates to a therapeutic agent and adiagnostic agent which are against frontotemporal lobar degeneration andtarget b-RAF described above. More specifically, the present inventionprovides the following.

<15> An agent for treating frontotemporal lobar degeneration, the agentcomprising a compound capable of suppressing an activity or expressionof b-RAF.<16> The agent according to <15>, wherein the compound is at least onecompound selected from the group consisting of PLX-4720, sorafenib,GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate, and LGX818.<17> The agent according to <16>, wherein the compound is vemurafenib.<18> An agent for diagnosing frontotemporal lobar degeneration, theagent comprising a compound having an activity of binding to b-RAF.

It should be noted that, in the present invention, “Alzheimer's disease”is a neurodegenerative disease also referred to as Alzheimer's dementiaor AD, and includes “familial Alzheimer's disease” and “inheritedAlzheimer's disease” attributable to gene mutation, and also “sporadicAlzheimer's disease” due to environmental factors such as lifestyle andstress. “Developing Alzheimer's disease” and related phrases mean anexpression of symptoms such as memory disorder, higher brain functiondisorders (aphasia, apraxia, agnosia, constructional apraxia), andchange in personality judged by clinical diagnosis, as well asappearance of atrophy in the brain judged by diagnostic imaging.“Affected with Alzheimer's disease” and related phrases mean to alsoinclude a state where the symptoms are not expressed, but a pathologicalchange peculiar to Alzheimer's disease (for example, amyloid βaggregation) occurs.

In addition, in the present invention, “frontotemporal lobardegeneration” is a non-Alzheimer's disease type neurodegenerativedisease also referred to as FTLD, by which atrophy occurs in the frontallobe and temporal lobe at the early stage, and atrophy occurs throughoutthe brain at the late stage. To be more specific, frontotemporal lobardegeneration includes three diseases classified according to clinicalcharacteristics: frontotemporal dementia (FTD), progressive nonfluentaphasia (PNFA), and semantic dementia (SD). Moreover, frontotemporallobar degeneration includes four diseases pathologically classified intoFTLD-Tau, FTLD-TDP, FTLD-UPS, and FTLD-FUS according to the type ofproteins accumulated as abnormal proteins in cells.

Further, “FTLD-Tau” is classified into “3R Tau” type, “4R Tau” type, and“3/4R Tau” type according to the number of microtubule-binding regionsrepeated in tau proteins predominantly accumulated in cells. Moreover,the “3R Tau” type includes FTLD with Pick bodies (Pick's disease), FTLDwith MAPT (microtubule-associated protein tau) gene mutation (FTLD-17),and the like. The “4R Tau” type includes corticobasal degeneration,progressive supranuclear palsy, multiple system tauopathy with dementia,argyrophilic grain dementia (argyrophilic grain disease), FTLD with MAPTgene mutation (FTLD-17), and the like. The “3/4R Tau” type includesdementia with neurofibrillary tangles, FTLD with MAPT gene mutation(FTLD-17), and the like. On the other hand, a FTLD group havingtau-negative, ubiquitin-positive inclusions is called “FTLD-U”, andincludes FTLD-TDP, FTLD-UPS and FTLD-FUS described above.

Among FTLD-U, “FTLD-TDP” means a TDP-43-positive disease. This diseaseincludes FTLD with PGRN (progranulin gene) mutation, sporadicFTLD-TDP/FTLD-U, FTLD with TARDBP (TDP-43 gene) mutation, FTLD with VCP(valosi-containing protein gene) mutation, FTLD linked to chromosome 9,and the like. Moreover, “FTLD-FUS” among FTLD-U means TDP-43-negative,FUS (fused in sarcoma)-positive disease. This disease includes neuronalintermediate filament inclusion disease, non-typical FTLD-U, basophilicinclusion body disease, FTLD with FUS mutation, and the like.

Further, “FTLD-UPS” is one of TDP-43-negative FTLD-U, This diseaseincludes FTLD with CHMP2B (charged multivesicular body protein 2B gene)mutation, and the like.

“Developing frontotemporal lobar degeneration” and related phrases meanan expression of symptoms such as memory disorder, higher brain functiondisorders (aphasia, apraxia, agnosia, constructional apraxia), andchange in personality judged by clinical diagnosis, as well asappearance of atrophy in the brain judged by diagnostic imaging.“Affected with frontotemporal lobar degeneration” and related phrasesmean to also include a state where the symptoms are not expressed, but apathological change peculiar to frontotemporal lobar degeneration (forexample, accumulation of the abnormal proteins in cells) occurs.

The “test subject” is a subject of the diagnosis method of the presentinvention, and includes not only bodies of animals including human, butalso body fluids, tissues, cells, and the like (for example,cerebrospinal fluid, cranial nerve tissues (particularly neurologicalbioptic tissues), blood, blood plasma, serous fluid, lymph, urine,saliva) isolated from the bodies.

The term “normal” in the normal subject means a state where the subjectis not affected with at least the disease (Alzheimer's disease orfrontotemporal lobar degeneration) to be targeted by the diagnosismethod of the present invention. Additionally, in the diagnosis methodof the present invention, a normal subject used as a comparison targetof a test subject is preferably the same gender as the test subject andsimilar in age.

Furthermore, in the present invention, “MARCKS” is a protein alsoreferred to as myristoylated alanine-rich C-kinase substrate. A typicalhuman-derived example thereof includes a protein specified under RefSeqID: NP_002347. Moreover, a typical example of a human-derived nucleicacid encoding MARCKS includes a nucleic acid containing a coding region(CDS) represented by RefSeq ID: NM_002358.

“Marcksl1” is a protein also referred to as MARCKS-like protein 1. Atypical human-derived example thereof includes a protein specified underRefSeq ID: NP_075385. Moreover, a typical example of a human-derivednucleic acid encoding MARCKS includes a nucleic acid containing a CDSrepresented by RefSeq ID: NM_023009.

“SRRM2” is a protein also referred to as SRm300/serine-argininerepetitive matrix protein 2. A typical human-derived example thereofincludes a protein specified under RefSeq ID: NP_057417. Moreover, atypical example of a human-derived nucleic acid encoding SRRM2 includesa nucleic acid containing a CDS represented by RefSeq ID: NM_016333.

“SPTA2” is a protein also referred to as α-II spectrin. Typicalhuman-derived examples thereof include a protein specified under RefSeqNP_001123910, a protein specified under RefSeq NP_001182461, and aprotein specified under RefSeq NP_003118. Moreover, typical examples ofa human-derived nucleic acid encoding SPTA2 include a nucleic acidcontaining a CDS represented by RefSeq NM_001130438, a nucleic acidcontaining a CDS represented by RefSeq NM_001195532, and a nucleic acidcontaining a CDS represented by RefSeq NM_003127.

“ADDB” is a protein also referred to as β adducin. Typical human-derivedexamples thereof include a protein specified under RefSeq NP_001171983,a protein specified under RefSeq NP_001171984, a protein specified underRefSeq NP_001608, a protein specified under RefSeq NP_059516, and aprotein specified under RefSeq NP_059522. Moreover, typical examples ofa human-derived nucleic acid encoding ADDB include a nucleic acidcontaining a CDS represented by RefSeq NM_001185054, a nucleic acidcontaining a CDS represented by RefSeq NM_001185055, a nucleic acidcontaining a CDS represented by RefSeq NM_001617, a nucleic acidcontaining a CDS represented by RefSeq NM_017482, and a nucleic acidcontaining a CDS represented by RefSeq NM_017488.

“NEUM” is a protein also referred to as neuromodulin or GAP43. Typicalhuman-derived examples thereof include a protein specified under RefSeqNP_00112353 and a protein specified under RefSeq NP_002036. Moreover,typical examples of a human-derived nucleic acid encoding NEUM include anucleic acid containing a CDS represented by RefSeq NM_001130064 and anucleic acid containing a CDS represented by RefSeq NM_002045.

“BASP1” is a protein also referred to as NAP-22 or CAP23. Typicalhuman-derived examples thereof include a protein specified under RefSeqNP_001258535 and a protein specified under RefSeq NP_006308. Moreover,typical examples of a human-derived nucleic acid encoding BASP1 includea nucleic acid containing a CDS represented by RefSeq NM_001271606 and anucleic acid containing a CDS represented by RefSeq NM_006317.

“SYT1” is a protein also referred to as synaptotagmin 1. Typicalhuman-derived examples thereof include a protein specified under RefSeqNP_00112927, a protein specified under RefSeq NP_001129278, and aprotein specified under RefSeq NP_005630. Moreover, typical examples ofa human-derived nucleic acid encoding SYT1 include a nucleic acidcontaining a CDS represented by RefSeq NM_001135805, a nucleic acidcontaining a CDS represented by RefSeq NM_001135806, and a nucleic acidcontaining a CDS represented by RefSeq NM_005639.

“G3P” is a protein also referred to as glyceraldehyde-3-phosphatedehydrogenase. Typical human-derived examples thereof include a proteinspecified under RefSeq NP_001243728 and a protein specified under RefSeqNP_002037. Moreover, typical examples of a human-derived nucleic acidencoding G3P include a nucleic acid containing a CDS represented byRefSeq NM_001256799 and a nucleic acid containing a CDS represented byRefSeq NM_002046.

“HS90A” is a protein also referred to as HSP90, HSP90α, or HSP86.Typical human-derived examples thereof include a protein specified underRefSeq NP_001017963 and a protein specified under RefSeq NP_005339.Moreover, typical examples of a human-derived nucleic acid encodingHS90A include a nucleic acid containing a CDS represented by RefSeqNM_001017963 and a nucleic acid containing a CDS represented by RefSeqNM_005348.

“CLH” is a protein also referred to as CLH1 or clathrin heavy chain 1. Atypical human-derived example thereof includes a protein specified underNP_004850. Moreover, a typical example of a human-derived nucleic acidencoding CLH includes a nucleic acid containing a CDS represented byNM_004859.

“NFH” is a protein also referred to as neurofilament heavy polypeptide.A typical human-derived example thereof includes a protein specifiedunder RefSeq NP_066554. Moreover, a typical example of a human-derivednucleic acid encoding NFH includes a nucleic acid containing a CDSrepresented by RefSeq NM_021076.

“NFL” is a protein also referred to as neurofilament light polypeptide.A typical human-derived example thereof includes a protein specifiedunder RefSeq NP_006149. Moreover, a typical example of a human-derivednucleic acid encoding NFL includes a nucleic acid containing a CDSrepresented by RefSeq NM_006158.

“GPRIN1” is a protein also referred to as G protein regulated inducer 1.Typical human-derived examples thereof include a protein specified underRefSeq NP_443131.2 and a protein specified under RefSeq XP_005265863.Moreover, typical examples of a human-derived nucleic acid encodingGPRIN1 include a nucleic acid containing a CDS represented by RefSeqNM_052899 and a nucleic acid containing a CDS represented by RefSeqXM_005265806.

“ACON” is a protein also referred to as aconitate hydratase. A typicalhuman-derived example thereof includes a protein specified under RefSeqNP_001089. Moreover, a typical example of a human-derived nucleic acidencoding ACON includes a nucleic acid containing a CDS represented byRefSeq NM_001098.

“ATPA” is a protein also referred to as ATP synthase subunit α, ATP5A1,ATP5A, ATP5AL2, or ATPM. Typical human-derived examples thereof includea protein specified under RefSeq NP_001001935, a protein specified underRefSeq NP_001001937, a protein specified under RefSeq NP_001244263, aprotein specified under RefSeq NP_001244264, and a protein specifiedunder RefSeq NP_004037. Moreover, typical examples of a human-derivednucleic acid encoding ATPA include a nucleic acid containing a CDSrepresented by RefSeq NM_001001935, a nucleic acid containing a CDSrepresented by RefSeq NM_001001937, a nucleic acid containing a CDSrepresented by RefSeq NM_001257334, a nucleic acid containing a CDSrepresented by RefSeq NM_001257335, and a nucleic acid containing a CDSrepresented by RefSeq NM_004046.

“ATPB” is a protein also referred to as ATP synthase subunit β, ATPMB,or ATPSB. A typical human-derived example thereof includes a proteinspecified under RefSeq NP_001677. Moreover, a typical example of ahuman-derived nucleic acid encoding ATPB includes a nucleic acidcontaining a CDS represented by RefSeq NM_001686.

Further, in the present invention, “PKC” is a protein also referred toas protein kinase C. Examples thereof include PKCβ, PKCα, PKCλ/ι(lambda/iota), PKCσ (delta), and PKCζ (zeta).

Typical examples of human-derived “PKCβ” include a protein specifiedunder RefSeq NP_002729 and a protein specified under RefSeq NP_997700.Moreover, a typical example of a human-derived nucleic acid encodingPKCβ includes a nucleic acid containing a CDS represented by RefSeqNM_002738 and a nucleic acid containing a CDS represented by RefSeqNM_212535.

A typical example of human-derived “PKCα” includes a protein specifiedunder RefSeq NP_002728. Moreover, a typical example of a human-derivednucleic acid encoding PKCα includes a nucleic acid containing a CDSrepresented by RefSeq NM_002737.

A typical example of human-derived “PKCλ/ι” includes a protein specifiedunder RefSeq NP_002731. Moreover, a typical example of a human-derivednucleic acid encoding PKCλ/ι includes a nucleic acid containing a CDSrepresented by RefSeq NM_002740.

Typical examples of human-derived “PKCσ” include a protein specifiedunder RefSeq NP_006245 and a protein specified under RefSeq NP_997704.Moreover, typical examples of a human-derived nucleic acid encoding PKCσinclude a nucleic acid containing a CDS represented by RefSeq NM_006254and a nucleic acid containing a CDS represented by RefSeq NM_212539.

Typical examples of human-derived “PKCζ” include a protein specifiedunder RefSeq NP_001028753, a protein specified under RefSeqNP_001028754, a protein specified under RefSeq NP_001229803, and aprotein specified under RefSeq NP_002735. Moreover, typical examples ofa human-derived nucleic acid encoding PKCζ include a nucleic acidcontaining a CDS represented by RefSeq NM_001033581, a nucleic acidcontaining a CDS represented by RefSeq NM_001033582, a nucleic acidcontaining a CDS represented by RefSeq NM_001242874, and a nucleic acidcontaining a CDS represented by RefSeq NM_002744.

“CaMK” is a protein also referred to as calmodulin kinase orcalmodulin-dependent protein kinase. Examples thereof include CaMKI,CaMKIIβ, CaMKIV, CaMKIIσ (delta), and CaMKIIα.

A typical example of human-derived “CaMKI” includes a protein specifiedunder RefSeq NP_003647. Moreover, a typical example of a human-derivednucleic acid encoding CaMKI includes a nucleic acid containing a CDSrepresented by RefSeq NM_003656.

Typical human-derived examples of “CaMKIIβ” include a protein specifiedunder RefSeq NP_001211, a protein specified under RefSeq NP_742075, aprotein specified under RefSeq NP_742076, a protein specified underRefSeq NP_742077, a protein specified under RefSeq NP_742078, a proteinspecified under RefSeq NP_742079, a protein specified under RefSeqNP_742080, a protein specified under RefSeq NP_742081, and a proteinspecified under RefSeq XP_005249918. Moreover, typical examples of ahuman-derived nucleic acid encoding CaMKIIβ include a nucleic acidcontaining a CDS represented by RefSeq NM_001220, a nucleic acidcontaining a CDS represented by RefSeq NM_172078, a nucleic acidcontaining a CDS represented by RefSeq NM_172079, a nucleic acidcontaining a CDS represented by RefSeq NM_172080, a nucleic acidcontaining a CDS represented by RefSeq NM_172081, a nucleic acidcontaining a CDS represented by RefSeq NM_172082, a nucleic acidcontaining a CDS represented by RefSeq NM_172083, a nucleic acidcontaining a CDS represented by RefSeq NM_172084, and a nucleic acidcontaining a CDS represented by RefSeq XM_005249861.

A typical example of human-derived “CaMKIV” includes a protein specifiedunder RefSeq NP_001735. Moreover, a typical example of a human-derivednucleic acid encoding CaMKIV includes a nucleic acid containing a CDSrepresented by RefSeq NM_001744.

Typical examples of human-derived “CaMKIIσ” include a protein specifiedunder RefSeq NP_001212, a protein specified under RefSeq NP_742112, aprotein specified under RefSeq NP_742113, a protein specified underRefSeq NP_742125, a protein specified under RefSeq NP_742126, a proteinspecified under RefSeq NP_742127, and a protein specified under RefSeqXP_005263312. Moreover, typical examples of a human-derived nucleic acidencoding CaMKIIσ include a nucleic acid containing a CDS represented byRefSeq NM_001221, a nucleic acid containing a CDS represented by RefSeqNM_172114, a nucleic acid containing a CDS represented by RefSeqNM_172115, a nucleic acid containing a CDS represented by RefSeqNM_172127, a nucleic acid containing a CDS represented by RefSeqNM_172128, a nucleic acid containing a CDS represented by RefSeqNM_172129, and a nucleic acid containing a CDS represented by RefSeqXM_005263255.

A typical example of human-derived “CaMKIIα” includes a proteinspecified under RefSeq NP_741960. Moreover, a typical example of ahuman-derived nucleic acid encoding CaMKIIα includes a nucleic acidcontaining a CDS represented by RefSeq NM_171825. “CSK” is a proteinalso referred to as casein kinase. Examples thereof include CSKIIα andCSKII subunit α.

Typical examples of human-derived “CSKIIα” include a protein specifiedunder RefSeq NP_001886, a protein specified under RefSeq NP_808227, anda protein specified under RefSeq NP_808228. Moreover, typical examplesof a human-derived nucleic acid encoding CSKIIα include a nucleic acidcontaining a CDS represented by RefSeq NM_001895, a nucleic acidcontaining a CDS represented by RefSeq NM_177559, and a nucleic acidcontaining a CDS represented by RefSeq NM_177560.

A typical example of human-derived “CSKII subunit α” includes a proteinspecified under RefSeq NP_001887. Moreover, a typical example of ahuman-derived nucleic acid encoding CSKII subunit α includes a nucleicacid containing a CDS represented by RefSeq NM_001896.

“Lyn” is a protein also referred to as Lyn tyrosine kinase. Typicalhuman-derived examples thereof include a protein specified under RefSeqNP_001104567, a protein specified under RefSeq NP_002341, a proteinspecified under RefSeq XP_005251289, and a protein specified underRefSeq XP_005251290. Moreover, typical examples of a human-derivednucleic acid encoding Lyn include a nucleic acid containing a CDSrepresented by RefSeq NM_001111097, a nucleic acid containing a CDSrepresented by RefSeq NM_002350, a nucleic acid containing a CDSrepresented by RefSeq XM_005251232, and a nucleic acid containing a CDSrepresented by RefSeq XM_005251233.

“b-RAF” is a protein also referred to as b-RAF serine/threonine kinase.A typical human-derived example thereof includes a protein specifiedunder RefSeq NP_004324. Moreover, a typical example of a human-derivednucleic acid encoding b-RAF includes a nucleic acid containing a CDSrepresented by RefSeq NM_004333.4.

Advantageous Effects of Invention

The present invention makes it possible to diagnose before the onset ofAlzheimer's disease, and further to provide agents and methods effectivefor treating the disease. Moreover, the present invention makes itpossible to diagnose frontotemporal lobar degeneration before the onset,and further to provide agents and methods effective for treating thedisease.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file of this patent contains at least onedrawing executed in color. Copies of this patent with color drawing(s)will be provided by the Patent and Trademark Office upon request andpayment of the necessary fee.

FIG. 1 is a diagram showing phosphoproteins which were identified by twodifferent approaches (hypothesis free approach and Aβ aggregation-linkedapproach) and whose expression amounts were enhanced commonly inmultiple Alzheimer's disease (AD) model mice. In the figure, “MARCS” and“MARCKSL1” respectively represent MARCKS and Marcksl1.

FIG. 2 is a schematic diagram showing a network constructed of the 17proteins shown in FIG. 1 . Note that the 17 proteins are proteins (ADcore proteins) revealed to have phosphorylation levels enhanced in thebrains affected with Alzheimer's disease identified in the presentinvention and to play central roles in a pathology of the disease.Moreover, in the figure, lines (edges) connecting the proteins (nodes)represent interactions therebetween.

FIG. 3 is a schematic diagram showing chronological changes inphosphorylations of kinases and substrate proteins thereof in apre-onset stage of Alzheimer's disease. To be more specific, it is shownthat: at the ages of 1 to 3 months, the phosphorylations of MARCKS,MARCKSL1, and SRRM2 in AD model mice (5×FAD mice) are remarkably high incomparison with those of the wild type; at the ages of 3 to 6 months,the phosphorylations of G3P, SYT1, SPTA2, ADDB, NEUM, BASP1, and HSP90Ain the AD model mice are remarkably high in comparison with those of thewild type; and at the age of 6 months or later, the phosphorylations ofCLH, NFH, NFL, and GPRIN1 in the AD model mice are remarkably high incomparison with those of the wild type. Note that, in the 12-month-oldAD model mice, no onset (such as abnormal behavior) is observed.Moreover, in the figure, lines (edges) connecting the proteins (nodes)represent interactions therebetween. In the figure, regarding “PKC”, seeJ Biol Chem., 1994, Vol. 269, No. 30, pp. 19462 to 19465 and JNeurochem., 1999, Vol. 73, Iss. 3, pp. 921 to 932. In addition,regarding “CSKII”, see J Biol Chem., 1993, Vol. 268, No. 9, pp. 6816 to6822.

FIG. 4 shows photograph for illustrating the result of administering akinase inhibitor (Go6976 or KN-93) or a Lyn kinase activator (MLR1023)into 5×FAD mice (12 weeks old), and observing dendritic spines/dendritesin layer 1 of the retrosplenial cortex 36 hours and 60 hours thereafter.Note that the observation results of administering DMSO (solvent alone)into 5×FAD mice and background mice thereof (B6/SJL (WT)) as controlsare also shown together.

FIG. 5 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine densities in the 5×FAD mice 36 hours and60 hours after Go6976, KN-93 or MLR1023 was administered. To be morespecific, the graphs show that clearly the number of protrusions wasdecreased in the 5×FAD mice, while the treatment with Go6976, KN-93, orMLR1023 recovered the decrease of spines in the 5×FAD mice. In thefigure, A shows the result of administering DMSO (solvent alone) intothe background (WT) mice, B shows the result of administering DMSO intothe 5×FAD mice, C shows the result of administering Go6976 into the5×FAD mice, D shows the result of administering MLR1023 into the 5×FADmice, and E shows the result of administering KN-93 into the 5×FAD mice.Moreover, each bar graph shows the average value +/− the standard error,and is provided with numerical values indicating the number of samplesin each administration group. One and two asterisks respectivelyindicate p<0.05 and p<0.01 Student's independent t-test (regarding therepresentations in the figure, the same shall apply to FIGS. 6, 8, 9,and 11 ).

FIG. 6 shows graphs for illustrating the result of quantitativelyanalyzing the spine type 36 hours and 60 hours after Go6976, KN-93, orMLR1023 was administered. To be more specific, the graphs show that,regardless of the morphological type, the spine densities were decreasedin the 5×FAD mice, and that the decreases were recovered by thetreatment with Go6976, KN-93, or MLR1023.

FIG. 7 shows photographs illustrating the spine formation andelimination in the 5×FAD mice 36 hours and 60 hours after Go6976, KN-93,or MLR1023 was administered. The arrow in the figure after 36 hours (36h) indicates the spine to be eliminated. The arrows in the figure after60 hours (60 h) indicate formed spines. Note that the observationresults of administering DMSO (solvent alone) into the 5×FAD mice andthe background mice (B6/SJL (WT)) as the controls are also showntogether.

FIG. 8 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine dynamics in the 5×FAD mice 36 hours and 60hours after Go6976, KN-93, or MLR1023 was administered. In the figure,the vertical axis represents the number of formed spines, eliminatedspines, or stably remaining spines per 100 μm of the dendritic shaft.

FIG. 9 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine dynamics in the 5×FAD mice 36 hours and 60hours after Go6976, KN-93, or MLR1023 was administered. In the figure,the vertical axis represents the relative percentage of formed spines,eliminated spines, or stably remaining spines.

FIG. 10 shows micrographs for illustrating the result of analyzing thecerebral cortexes of WT/DMSO (the background mice treated with DMSOalone), 5×FAD/DMSO (the 5×FAD mice treated with DMSO alone), 5×FAD/Go(the 5×FAD mice treated with Go6976), 5×FAD/MLR (the 5×FAD mice treatedwith MLR1023), and 5×FAD/KN-93 (the 5×FAD mice treated with KN-93) byimmunohistological staining using antibodies against activated PKCβ(PKCβ pT642), activated PKCδ (PKCδ pS643/676), and activated CamKII(CamKII pT286).

FIG. 11 shows graphs for illustrating the result of quantitativelyanalyzing signals from each activated kinase in neuronal somas orneuropils of the five types of mice shown in FIG. 10 .

FIG. 12 shows photographs for illustrating the result of injecting alentiviral vector encoding shRNA against MARCKS into layer 1 of theretrosplenial cortex, and observing dendritic spines/dendrites at thesite 4 days and 5 days later. Note that the observation results ofinjecting scrambled shRNA into 5×FAD mice and background mice thereof(B6/SJL (WT)) as controls are also shown together.

FIG. 13 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine densities in the 5×FAD mice 4 days and 5days after the shRNA against MARCKS was injected. In the figure, A showsthe result of injecting the scrambled shRNA into the background (WT)mice, B shows the result of injecting the scrambled shRNA into the 5×FADmice, and C shows the result of injecting the shRNA against MARCKS intothe 5×FAD mice. Moreover, each bar graph shows the average value +/− thestandard error, and is provided with numerical values (n=4) indicatingthe number of samples in each injection group. One and two asterisksrespectively indicate p<0.05 and p<0.01 in Student's independent t-test(regarding the representations in the figure, the same shall apply toFIGS. 14, 15, 17 , and 18).

FIG. 14 shows graphs for illustrating the result of quantitativelyanalyzing the spine type 4 days after the shRNA against MARCKS wasinjected.

FIG. 15 shows graphs for illustrating the result of quantitativelyanalyzing the spine type 5 days after the shRNA against MARCKS wasinjected.

FIG. 16 shows photographs illustrating the spine formation andelimination in the 5×FAD mice 4 days and 5 days after the shRNA againstMARCKS was injected. In the figure, the arrow provided to theobservation result after 4 days (4 d) indicates the spine to beeliminated, while the other arrows indicate formed spines. Note that theobservation result of injecting the scrambled shRNA into the 5×FAD miceand the background mice (B6/SJL (WT)) as the controls are also showntogether.

FIG. 17 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine dynamics in the 5×FAD mice in which theshRNA against MARCKS was injected. In the figure, the vertical axisrepresents the number of formed spines, eliminated spines, or stablyremaining spines per 100 μm of the dendritic shaft.

FIG. 18 shows graphs for illustrating the result of quantitativelyanalyzing the dendritic spine dynamics in the 5×FAD mice in which theshRNA against MARCKS was injected. In the figure, the vertical axisrepresents the relative percentage of formed spines, eliminated spines,or stably remaining spines.

FIG. 19 shows photographs for illustrating the result of treating mouseprimary cortical nerve cells (E18) with a 10-μM amyloid β protein(Aβ1-42) in a medium for 6 hours in the presence or absence of a b-rafkinase inhibitor, followed by analysis by western blotting using ananti-phosphorylated tau antibody. Note that, as the b-raf kinaseinhibitor, PLX-4720 (PLX), sorafenib (Sor), and GDC-0879 (GDC) wereused, each of which was added to the medium at the concentration of 1 μMor 10 μM.

FIG. 20 is a graph for illustrating the result of quantitativelyanalyzing bands of the western blot shown in FIG. 19 . In Student'sindependent t-test, one asterisk indicates p<0.05 (n=3).

FIG. 21 is a schematic diagram showing a protocol for analyzing thetherapeutic effect of a b-raf inhibitor on the behavioral phenotype ofFTLD model mice.

FIG. 22 shows graphs for illustrating the result of providing a b-rafinhibitor (vemurafenib) to FTLD model mice (PGRN-KI mice), andevaluating the behavior of these mice by a Morris water maze test. Inthe figure, the numbers shown in bars of the graphs indicate the numbersof mice analyzed in each test. Moreover, “−” shows the result (negativecontrol) of mice to which PBS was administered in place of vemurafenib.

FIG. 23 is a graph for illustrating the result of providing vemurafenibto the PGRN-KI mice, and evaluating the behavior of these mice by afear-conditioning test (the representations in the figure are the sameas FIG. 22 ).

FIG. 24 is a schematic diagram showing a protocol analyzing thetherapeutic effect of a TNF signal transduction inhibitor on thebehavioral phenotype of the FTLD model mice.

FIG. 25 shows graphs for illustrating the result of providing a TNFsignal transduction inhibitor (thalidomide) to the FTLD model mice(PGRN-KI mice), and evaluating the behavior of these mice by the Morriswater maze test. In the figure, the numbers shown in bars of the graphsindicate the numbers of mice analyzed in each test. Moreover, “−” showsthe result (negative control) of mice to which PBS was administered inplace of thalidomide.

FIG. 26 is a graph for illustrating the result of providing thalidomideto the PGRN-KI mice, and evaluating the behavior of these mice by thefear-conditioning test (the representations in the figure are the sameas FIG. 25 ).

FIG. 27 is a figure for illustrating the result of western blot analysison the cerebral cortexes of wild-type mice (WT) and the PGRN-KI mice towhich vemurafenib was administered. In the figure, the upper panel showsphotographs for illustrating the western blot analysis result.“Anti-p-Braf” and “Anti-p-PKC” respectively show the analysis result ofphosphorylated b-RAF protein and the analysis result of phosphorylatedPKC protein. “Anti-GAPDH” shows the result of detecting a GAPDH proteinas an internal standard. Moreover, in the figure, the lower two panelsare graphs for illustrating the western blot analysis result: the leftside shows the relative value of the phosphorylated b-RAF protein amountbased on the GAPDH amount, and the right side shows the relative valueof the phosphorylated PKC protein amount based on the GAPDH amount. Thesignificant differences were evaluated based on the P-value calculatedby Student's independent t-test.

FIG. 28 is a figure for illustrating the result of western blot analysison the cerebral cortexes of the PGRN-KI mice to which thalidomide wasadministered. In the figure, the left panel shows photographs forillustrating the western blot analysis result. “Anti-p-Braf”,“Anti-Braf”, and “Anti-p-PKC” respectively show the analysis result ofphosphorylated b-raf protein, the analysis result of b-raf protein, andthe analysis result of phosphorylated PKC protein. “Anti-GAPDH” showsthe result of detecting a GAPDH protein as an internal standard.Moreover, in the figure, the right panel shows graphs for illustratingthe western blot analysis result. “p-B-raf/Braf” shows the relativevalue of the phosphorylated b-raf protein amount based on a total b-rafprotein amount, “p-B-raf/GAPDH” shows the relative value of thephosphorylated b-raf protein amount based on the GAPDH amount, and“p-PKC/GAPDH” shows the relative value of the phosphorylated PKC proteinamount based on the GAPDH amount. Further, in the figure, one asteriskindicates p<0.05 in Student's independent t-test, and two asterisksindicate p<0.01 in Student's independent t-test.

FIG. 29 is a figure for illustrating the result of spine static analysison the retrosplenial cortexes (RSD) of the FTLD model mice (PGRN-KImice) and wild-type mice (WT). In the figure, the left panel showsphotographs of spines observed with a two-photon microscope. In thefigure, graphs in the right panel show the number of spines (the numberof spines per 1 μm of the dendrite), spine length, spine head diameter,and spine volume measured by the two-photon microscope observation.Moreover, in each graph, the left bar shows the observation result ofthe wild-type mice, and the right bar shows the observation result ofthe PGRN-KI mice. In the figure, two asterisks indicate p<0.01 inStudent's independent t-test.

FIG. 30 is a figure for illustrating the result of spine dynamicanalysis on the retrosplenial cortexes (RSD) of the FTLD model mice(PGRN-KI mice) and wild-type mice (WT). In the figure, the left panelshows photographs of spines observed with a two-photon microscope. Inthe photographs, the upward arrows indicate spines to be eliminated, andthe downward arrows indicate produced spines. The right panel shows thenumber of spines produced, the number of spines eliminated, and thenumber of spines stably remaining detected by the two-photon microscopeobservation. Moreover, in each graph, the left bar shows the observationresult of the wild-type mice, and the right bar shows the observationresult of the PGRN-KI mice.

FIG. 31 is a figure for illustrating the result of spine static analysison the retrosplenial cortexes (RSD) of the PGRN-KI mice (KI) to whichthe b-raf inhibitor (vemurafenib) or PBS was administered. In thefigure, the left panel shows photographs of spines observed with atwo-photon microscope. In the figure, graphs in the right panel show thenumber of spines (the number of spines per 1 μm of the dendrite), spinelength, spine head diameter, and spine volume measured by the two-photonmicroscope observation. Moreover, in each graph, the left bar shows theobservation result of the PBS-administered PGRN-KI mice (KI+PBS), andthe right bar shows the observation result of thevemurafenib-administered PGRN-KI mice (KI+vemurafenib). In the figure,one asterisk indicates p<0.05 in Student's independent t-test.

FIG. 32 is a figure for illustrating the result of spine static analysison the retrosplenial cortexes (RSD) of the PGRN-KI mice (KI) in whichshRNA against tau (Sh-Tau) or scrambled shRNA (Sh-scrambled) wasinjected. In the figure, the left panel shows photographs of spinesobserved with a two-photon microscope. In the figure, graphs in theright panel show the number of spines (the number of spines per 1 μm ofthe dendrite), spine length, spine head diameter, and spine volumemeasured by the two-photon microscope observation. Moreover, in eachgraph, the left bar shows the observation result of the scrambledshRNA-injected PGRN-KI mice (KI+Sh-scrambled), and the right bar showsthe observation result of the Sh-Tau-injected PGRN-KI mice (KI+Sh-Tau).In the figure, one asterisk indicates p<0.05 in Student's independentt-test.

FIG. 33 shows graphs for illustrating the result of spine dynamicsanalysis on the retrosplenial cortexes of the FTLD model mice (PGRN-KImice). In the figure, the left graph shows the result of administeringvemurafenib to the PGRN-KI mice (the number of analyses: three mice, theleft bars in the graph) or the result of administering PBS to thePGRN-KI mice (the number of analyses: four mice, the right bars in thegraph). In the figure, the right graph shows the result of injectingscrambled shRNA into the PGRN-KI mice (the number of analyses: fourmice, the left bars in the graph) or the result of injecting shRNAagainst tau into the PGRN-KI mice (the number of analyses: four mice,the right bars in the graph).

FIG. 34 shows the comparison results between 14 proteins selected fromthe AD model mice by the hypothesis free approach and phosphoproteinswhich changed in the Tau model mice. As a result, it was revealed that10 phosphoproteins (ADDB, NFH, NFL, SPTA2, BASP1, CLH, MARCS, NEUM,SRRM2, and Marcksl1) were commonly changed between the AD model mice andthe Tau model mice.

DESCRIPTION OF EMBODIMENTS

<Method 1 for Diagnosing Alzheimer's Disease>

As will be described later in Examples, it has been revealed thatphosphorylations of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB arecommonly enhanced in multiple Alzheimer's disease model mice before theonset of the disease. Thus, the present invention provides a method fordiagnosing Alzheimer's disease based on the phosphorylation of theseproteins, the method comprising the following the steps (i) to (iii):

(i) a step of detecting, in a test subject, a phosphorylation of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB;

(ii) a step of comparing the phosphorylation with a phosphorylation of asubstrate protein in a normal subject; and

(iii) a step of determining that the test subject is affected withAlzheimer's disease or has a risk of developing Alzheimer's disease ifthe phosphorylation of the substrate protein in the test subject ishigher than the phosphorylation of the substrate protein in the normalsubject as a result of the comparison.

In this diagnosis method, the substrate proteins such as MARCKS are notlimited respectively to the proteins having the amino acid sequenceslisted as the typical examples described above, and naturally-occurringmutants thereof can also be targeted. Moreover, in a case where multiplesites (amino acid residues) are phosphorylated in one substrate protein,the phosphorylation of at least one site of the protein should bedetected. Nevertheless, from the viewpoint of further increasing thediagnosis precision, it is preferable to detect all the phosphorylationsites of the protein.

In the case of human, examples of the phosphorylation site in MARCKS tobe detected by the present invention include serine at position 26,serine at position 27, serine at position 29, serine at position 118,serine at position 128, serine at position 131, serine at position 132,serine at position 134, serine at position 135, serine at position 145,serine at position 147, threonine at position 150, serine at position170, and serine at position 322. Examples thereof in Marcksl1 includeserine at position 22, threonine at position 85, serine at position 104,threonine at position 148, serine at position 151, serine at position180, and serine at position 184. Examples thereof in SRRM2 includeserine at position 1102, serine at position 1320, serine at position1348, serine at position 1383, serine at position 1403, serine atposition 1404, serine at position 2398, serine at position 2132, serineat position 2449, serine at position 2581, threonine at position 1492,and threonine at position 2397. Examples thereof in SPTA2 include serineat position 1031 and serine at position 1217. Examples thereof in ADDBinclude serine at position 60, serine at position 62, serine at position532, serine at position 592, serine at position 600, serine at position617, serine at position 693, and serine at position 701. Examplesthereof in NEUM include serine at position 151, threonine at position181, and serine at position 203. Examples thereof in BASP1 includethreonine at position 31, threonine at position 36, serine at position132, serine at position 195, and serine at position 219. Examplesthereof in SYT1 include threonine at position 126 and threonine atposition 129. Examples thereof in G3P include threonine at position 184and threonine at position 211. Examples thereof in HS90A include serineat position 231 and serine at position 263. Examples thereof in NFHinclude serine at position 503, serine at position 540, serine atposition 660, serine at position 730, serine at position 769, serine atposition 801, and serine at position 828. An example thereof in NFLincludes serine at position 472. Examples thereof in GPRIN1 includeserine at position 776, serine at position 799, serine at position 850,serine at position 853, and threonine at position 877.

Note that these phosphorylation sites are sites in Alzheimer's diseasemodel mice where phosphorylation levels were changed and identified inExample 5 to be described later, and converted to corresponding humansites (regarding the correspondence between human and mouse at eachphosphorylation site, see PhosphoSite Plus(www.phosphosite.org/homeAction.do)). Additionally, in the presentinvention, the term “phosphorylation site” means a site having at least3 amino acids including one amino acid before and one amino acid after aphosphorylated amino acid in a phosphorylated protein such as thesubstrate protein.

In the diagnosis method, in a case where a test subject whose substrateprotein phosphorylation is to be detected is a specimen (such as bodyfluid, tissue, cell) isolated from a body of an animal including human,that is, where the diagnosis method is an in vitro method, an example ofthe detection method in the step (i) includes a mass spectrometry methodas will be described later in Examples, that is, a method in whichphosphopolypeptides are extracted from the specimen, labeled, andanalyzed by 2D LC MS/MS. Such a detection by a mass spectrometry methodis preferable from the viewpoint that multiple phosphorylations ofmultiple substrate proteins can be comprehensively detected,consequently further increasing the diagnosis precision.

Moreover, the in vitro method includes a detection method using anantibody capable of specifically binding to a phosphorylation site of asubstrate protein, for example, immunohistochemical staining,immunoelectron microscopy, and immunoassays (such as enzyme immunoassay(ELISA, EIA), fluorescent immunoassay, radioimmunoassay (RIA),immunochromatography, and western blot method). Further, the examplealso includes a method utilizing a detector (for example, BIAcore(manufactured by GE Healthcare)) based on the surface plasmon resonancephenomenon using a thin metal film on which a compound capable ofspecifically binding to a phosphorylation site of a substrate protein isimmobilized. Regarding the antibody and the compound, see thedescription of <Diagnostic Agent against Alzheimer's Disease> to bedescribed later.

Meanwhile, in the diagnosis method, in a case where a test subject whosesubstrate protein phosphorylation is to be detected is a body of ananimal including human, that is, where the diagnosis method is an invivo method, examples of the detection method in the step (i) includebioimaging techniques (computerized axial tomographies (CAT, CT),magnetic resonance imaging (MRI), positron emission tomography (PET),single-photon emission computed tomography (SPECT)). More concretely,the detection can be performed with reference to the techniquesdescribed in International Application Japanese-Phase Publication Nos.2004-513123, 2004-530408, and 2002-514610, Japanese Unexamined PatentApplication Publication No. 2011-95273, International ApplicationJapanese-Phase Publication Nos. 2001-527509, Hei 9-501419, Hei 9-505799,and Hei 8-509226. Nevertheless, the embodiment of the diagnosis methodof the present invention is not limited thereto.

In the bioimaging techniques, a compound capable of specifically bindingto a phosphorylation site of a substrate protein is introduced into thebody of a test subject. The introduction method is not particularlylimited, and examples thereof include intravenous administration,intraarterial administration, intraperitoneal administration,subcutaneous administration, intradermal administration,tracheobronchial administration, rectal administration and intramuscularadministration, administration by transfusion, and direct administrationinto a target site (such as brain). The direct administration into atarget site can be achieved by employing, for example, cannula(catheter), surgical incision, or the like. Regarding the compound, seethe description of <Diagnostic Agent against Alzheimer's Disease> to bedescribed later.

In addition, as will be described later in Examples, the substrateproteins to be detected in the method for diagnosing Alzheimer's diseaseare classified into three according to the pattern of the chronologicalchange in phosphorylation. To be more specific, examples of thesubstrate protein whose phosphorylation is enhanced the most at aninitial phase of a pre-onset stage of Alzheimer's disease includeMARCKS, Marcksl1, and SRRM2; examples of the substrate protein whosephosphorylation is enhanced the most at a mid phase of the pre-onsetstage of Alzheimer's disease include SPTA2, ADDB, NEUM, BASP1, SYT1,G3P, and HS90A; and examples of the substrate protein whosephosphorylation is enhanced the most at a late phase of the pre-onsetstage of Alzheimer's disease include CLH, NFH, NFL, and GPRIN1. Thus, inthe step (iii), if the phosphorylations of at least two substrateproteins among MARCKS, Marcksl1, and SRRM2 (more preferably thephosphorylations of all the three substrate proteins) are higher thanthose in a normal subject, the test subject can be determined to be atthe initial phase before the onset of Alzheimer's disease. Moreover, ifthe phosphorylations of at least two substrate proteins among SPTA2,ADDB, NEUM, BASP1, SYT1, G3P, and HS90A (more preferably thephosphorylations of three substrate proteins, the phosphorylations offour substrate proteins, the phosphorylations of five substrateproteins, furthermore preferably the phosphorylations of six substrateproteins, and particularly preferably the phosphorylations of all theseven substrate proteins) are higher than those in a normal subject, thetest subject can be determined to be at the mid phase before the onsetof Alzheimer's disease. Further, if the phosphorylations of at least twosubstrate proteins among CLH, NFH, NFL, and GPRIN1 (more preferably thephosphorylations of three substrate proteins, furthermore preferably thephosphorylations of all the four substrate proteins) are higher thanthose in a normal subject, the test subject can be determined to be atthe late phase before the onset of Alzheimer's disease.

<Method 2 for Diagnosing Alzheimer's Disease>

In addition, as will be described later in Examples, it has also beenrevealed that kinase proteins which phosphorylate the substrate proteinssuch as MARCKS are activated in the Alzheimer's disease model micebefore the onset of the disease. Thus, the present invention alsoprovides, as a second embodiment of the method for diagnosingAlzheimer's disease, a method comprising the following the steps (i) to(iii):

(i) a step of detecting an activity or expression of a kinase protein ina test subject;

(ii) a step of comparing the activity or expression with an activity orexpression of a kinase protein in a normal subject; and

(iii) a step of determining that the test subject is affected withAlzheimer's disease or has a risk of developing Alzheimer's disease ifthe activity or expression of the kinase protein in the test subject ishigher than the activity or expression of the kinase protein in thenormal subject as a result of the comparison, wherein

the kinase protein is at least one kinase protein selected from thegroup consisting of PKC, CaMK, CSK, Lyn, and b-RAF.

In this diagnosis method, the kinase proteins such as PKC are notlimited respectively to the proteins having the amino acid sequenceslisted as the typical examples described above, and naturally-occurringmutants thereof can also be targeted. Moreover, the “activity” of thekinase proteins to be detected means an activity (kinase activity) ofdirectly or indirectly phosphorylating the substrate protein. Further,since a kinase activity correlates with an amount of a kinase proteinexpressed, particularly an amount of an activated kinase proteinexpressed, the amount of a kinase protein expressed, preferably theamount of an activated kinase protein expressed, can also be the targetof the detection by the diagnosis method in place of the kinaseactivity.

In the present invention, the “activated kinase protein” means a kinaseprotein in a state where the kinase protein is capable ofphosphorylating the substrate protein. An example thereof includes aphosphorylated kinase protein. More concretely, the examples of theactivated kinase protein include PKCβ having threonine at position 642phosphorylated, PKCα having threonine at position 638 phosphorylated,PKCλ/ι having threonine at position 403 phosphorylated, PKCδ havingserine at position 643 phosphorylated, PKCζ having threonine at position410 phosphorylated, PKCζ having tyrosine at position 417 phosphorylated,CaMKI having serine at position 177 phosphorylated, CaMKIIβ havingthreonine at position 287 phosphorylated, CaMKIV having threonine atposition 200 phosphorylated, CaMKIIσ having threonine at position 287phosphorylated, CaMKIIα having threonine at position 286 phosphorylated,CSKIIα having threonine at position 360 phosphorylated, CSKIIα havingserine at position 362 phosphorylated, Lyn having tyrosine at position397 phosphorylated, b-RAF having serine at position 365 phosphorylated,b-RAF having serine at position 446 phosphorylated, b-RAF having serineat position 579 phosphorylated, b-RAF having threonine at position 599phosphorylated, b-RAF having serine at position 602 phosphorylated,b-RAF having serine at position 729 phosphorylated, and b-RAF havingserine at position 732 phosphorylated.

In the case where the diagnosis method is an in vitro method, thedetection of the activity can be performed, for example, by adding asubstrate and a radiolabeled phosphate to the specimen or a proteinliquid extract thereof, and detecting the incorporation of the phosphateinto the substrate. The incorporation of the phosphate into thesubstrate can be detected with a scintillation counter, byautoradiography, or other means. Alternatively, without using aradioactive label, the activity can also be detected by treating thesubstrate with the specimen or a protein liquid extract thereof, anddetecting an increase in the molecular weight of the substrate after thetreatment. The detection of an increase in the molecular weight can beperformed, for example, by detecting a change in mobility of thesubstrate in polyacrylamide gel electrophoresis. Further, thepolyacrylamide gel after the electrophoresis may be transferred to amembrane such as PVDF for the detection by a western blot method usingan antibody capable of specifically binding to a phosphorylation site ofthe substrate. Note that examples of the substrate include knownsubstrate proteins of the targeted kinase proteins, and partial peptidesthereof containing a site to be phosphorylated (phosphorylated site).

Examples of the known substrate proteins of PKC include MARCKS,Marcksl1, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, and HSP90A. Moreconcretely, examples of the substrate proteins of PKCβ include SPTA2,MARCKS, and NEUM; examples of the substrate protein of PKCα includeMARCKS and HSP90A; an example of the substrate protein of PKCζ includesMarcksl1; examples of the substrate protein of PKCλ/I include G3P andHSP90A; and examples of the substrate protein of PKCδ include ADDB,NEUM, and HSP90A.

Examples of the known substrate proteins of CaMK include SPTA2 and G3P.More concretely, an example of the substrate protein of CaMKI includesG3P; an example of the substrate protein of CaMKIIβ includes G3P; anexample of the substrate protein of CaMKIV includes G3P; and an exampleof the substrate protein of CaMKIIδ includes SPTA2.

Examples of the known substrate proteins of CSK include NEUM, SYT1, andHSP90A. More concretely, an example of the substrate protein of CSKIIsubunit α includes HSP90A; and examples of the substrate protein ofCSKIIα include NEUM and HSP90A.

An example of the known substrate protein of Lyn includes G3P. Moreover,examples of the substrate protein of b-RAF include MEK1, ERK1, and tau.

Moreover, in the in vitro method, examples of the method for detectingthe expression amount include, as in the case of the detection ofsubstrate protein phosphorylation described above, a mass spectrometrymethod, a detection method using an antibody capable of specificallybinding to a kinase protein (preferably, activated kinase protein), anda method utilizing a detector based on the surface plasmon resonancephenomenon using a thin metal film on which a compound capable ofspecifically binding to a kinase protein (preferably, activated kinaseprotein) is immobilized. Regarding the antibody and the compound, seethe description of <Diagnostic Agent against Alzheimer's Disease> to bedescribed later.

In the case where the diagnosis method is an in vivo method, thedetection of the activity can be performed by detecting the substrateprotein phosphorylation attributable to the activity. To be morespecific, bioimaging techniques aiming at the detection of substrateprotein phosphorylation described above can be suitably used.

Moreover, in the case where the diagnosis method is an in vivo method,the detection of the expression amount can be performed as in the caseof the detection of substrate protein phosphorylation described above,for example, by utilizing bioimaging techniques using a compound capableof specifically binding to a kinase protein (preferably, activatedkinase protein). Regarding the compound, see the description of<Diagnostic Agent against Alzheimer's Disease> to be described later.

Hereinabove, preferred embodiments of the diagnosis method of thepresent invention have been described. In addition, such a diagnosis isnormally conducted by a doctor (including one instructed by a doctor).The data on the phosphorylation of the substrate protein or the activityor expression of the kinase protein in the test subject obtained by thediagnosis method of the present invention is useful in the diagnosis bya doctor. Thus, the method of the present invention can also bedescribed as a method for collecting and presenting such data useful ina diagnosis by a doctor.

Moreover, the present invention makes it possible to determine that oneis affected with Alzheimer's disease or has a risk of developingAlzheimer's disease. In this manner, enabling judgment of Alzheimer'sdisease affection or the like at an early stage leads to an expectationthat treatment methods for suppressing a pathology of Alzheimer'sdisease (immunotherapy, a method for administering an agent forsuppressing a pathology of Alzheimer's disease) will be effective.

Thus, the present invention also makes it possible to provide a methodfor treating Alzheimer's disease, the method comprising: a step ofadministering an agent for suppressing a pathology of Alzheimer'sdisease to a test subject determined to be affected with Alzheimer'sdisease or have a risk of developing Alzheimer's disease by the methodfor diagnosing Alzheimer's disease of the present invention, and/or astep of conducting an immunotherapy for the test subject.

Examples of the immunotherapy include active immunotherapy (vaccinetherapy) using a partial peptide of amyloid β to suppress amyloid βaggregation, and passive immunotherapy in which an antibody againstamyloid β is administered.

Additionally, examples of the agent administered to suppress a pathologyof Alzheimer's disease include agents for suppressing amyloid βproduction (such as γ-secretase modulators (GSM), γ-secretase modulatorinhibitors (GSI), nonsteroidal anti-inflammatory drugs), agents forsuppressing amyloid β aggregation (such as curcumin, polysulfuric acidcompound, clioquinol), agents for suppressing tau aggregation (such asaminothienopyridazine, cyanine dye, methylene blue), neuroprotectivedrugs (such as dimebon), cholinesterase inhibitors (such as donepezil),acetylcholinesterase inhibitors (such as galantamine), and NMDAglutamate receptor inhibitors (such as memantine).

Further, as will be described later, it is also possible to suitablyuse, as the agent for suppressing a pathology of Alzheimer's disease, anagent for treating Alzheimer's disease, the agent comprising any one of:a compound capable of suppressing a phosphorylation of at least onesubstrate protein selected from the group consisting of MARCKS,Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH,NFL, GPRIN1, ACON, ATPA, and ATPB; a compound capable of suppressing anactivity or expression of at least one kinase protein selected from thegroup consisting of PKC, CaMK, CSK, Lyn, and b-RAF; a compound capableof activating Lyn; and a compound capable of suppressing a binding of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinaseprotein selected from the group consisting of PKC, CaMK, CSK, Lyn, andb-RAF.

<Diagnostic Agent Against Alzheimer's Disease>

As described above, it has been revealed that the phosphorylations ofMARCKS and the like are commonly enhanced in the multiple Alzheimer'sdisease model mice before the onset of the disease. Thus, the presentinvention provides an agent for diagnosing Alzheimer's disease, theagent comprising a compound having an activity of binding to aphosphorylation site of at least one substrate protein selected from thegroup consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB(hereinafter, the agent for diagnosing Alzheimer's disease may also bereferred to as “Alzheimer's disease diagnostic agent”).

As described above, it has been revealed that the kinase proteins whichphosphorylate the substrate proteins such as MARCKS are also activatedin the Alzheimer's disease model mice before the onset of the disease.Thus, the present invention provides an agent for diagnosing Alzheimer'sdisease, the agent comprising a compound having an activity of bindingto at least one kinase protein selected from the group consisting ofPKC, CaMK, CSK, Lyn, and b-RAF. Further, an example of a preferredembodiment of the agent includes an agent for diagnosing Alzheimer'sdisease, the agent comprising a compound having an activity of bindingto at least one activated kinase protein selected from the groupconsisting of PKC, CaMK, CSK, Lyn, and b-RAF.

As in the diagnosis method described above, the substrate proteins andthe kinase proteins targeted by the diagnostic agents of the presentinvention are not limited respectively to the proteins having the aminoacid sequences listed as the typical examples described above, andnaturally-occurring mutants thereof can also be targeted.

The “compound having an activity of binding to the phosphorylation siteof the substrate protein” and the “compound having an activity ofbinding to the kinase protein” are not particularly limited, and may beknown compounds or may be ones identified by screening to be describedlater. Examples of such compounds include antibodies capable of bindingto the phosphorylation site of the substrate protein or the kinaseprotein, and low-molecular-weight compounds capable of binding to thephosphorylation site of the substrate protein or the kinase protein.

An “antibody” in the present invention may be a polyclonal antibody, amonoclonal antibody, or a functional fragment of an antibody. Theantibody includes all classes and subclasses of immunoglobulins. The“functional fragment” of an antibody means a part (partial fragment) ofan antibody and capable of specifically recognizing an antigen thereof.Concretely, examples thereof include Fab, Fab′, F(ab′)2, a variableregion fragment (Fv), a disulfide bonded Fv, a single chain Fv (scFv), asc(Fv)2, a diabody, a polyspecific antibody, polymers thereof, and thelike. Moreover, the antibody includes a chimeric antibody, a humanizedantibody, a human antibody, and functional fragments of theseantibodies. Further, the amino acid sequences of these antibodies mayundergo alteration, modification, or the like as necessary. Thoseskilled in the art can prepare such antibodies as appropriate by knownantibody preparation methods. Furthermore, in a case where the agentsfor diagnosing Alzheimer's disease of the present invention or agentsfor treating the disease to be described later are to be introduced intohuman, preferable among these antibodies are a humanized antibody, ahuman antibody, and functional fragments of these antibodies, from theviewpoint that an immunoreaction hardly occurs with the introducedantibody.

In addition, the “compound having an activity of binding to thephosphorylation site of the substrate protein” and the “compound havingan activity of binding to the kinase protein” preferably have a labelingsubstance bound thereto for the detection by the above-describeddetection methods using an antibody, bioimaging techniques, and thelike. The labeling substance is selected as appropriate in accordancewith the type of the detection method employed and the like. Examplesthereof include radioactive labeling substances, fluorescent labelingsubstances, paramagnetic labeling substances, superparamagnetic labelingsubstances, and enzyme labeling substances. Moreover, such labelingsubstances may be bound to the molecules directly or indirectly.Examples of the indirect binding include bindings utilizing a secondaryantibody to which a labeling substance is bound, or a polymer (such asProtein A, Protein B) to which a labeling substance is bound.

The agents of the present invention may comprise, in addition to thecompounds, other pharmacologically acceptable ingredients. Examples ofsuch other ingredients include a carrier, an excipient, a disintegrator,a buffer, an emulsifier, a suspension, a stabilizer, a preservative, anantiseptic, and a physiological salt. As the excipient, lactose, starch,sorbitol, D-mannitol, white sugar, or the like can be used. As thedisintegrator, starch, carboxymethyl cellulose, calcium carbonate, orthe like can be used. As the buffer, a phosphate, a citrate, an acetate,or the like can be used. As the emulsifier, gum arabic, sodium alginate,traganth, or the like can be used. As the suspension, glycerylmonostearate, aluminium monostearate, methyl cellulose, carboxymethylcellulose, hydroxymethyl cellulose, sodium lauryl sulfate, or the likecan be used. As the stabilizer, propylene glycol, diethyltin sulfite,ascorbic acid, or the like can be used. As the preservative, phenol,benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, orthe like can be used. As the antiseptic, sodium azide, benzalkoniumchloride, para-oxybenzoic acid, chlorobutanol, or the like can be used.

When the diagnostic agents of the present invention are used in vivo,the administration method into the body of a test subject is asdescribed above in the description of <Method for diagnosing Alzheimer'sdisease>. Moreover, those skilled in the art can adjust an amount of thediagnostic agents of the present invention administered and the numberof administrations as appropriate depending on the type of thecompounds, the body weight of a test subject, and the like. The numberof administrations can be adjusted as appropriate depending on theadministration amount, the administration route, and the like.

A product of the diagnostic agents of the present invention or a manualthereof may be provided with an indication stating that the product isused for diagnosing the target disease. Herein, “a product or a manualprovided with an indication” means that the indication is provided to amain body, a container, a package, or the like of the product, or theindication is provided to a manual, a package insert, an advertisement,other printed matters, or the like in which information on the productis disclosed.

<Screening Method for Alzheimer's Disease Diagnostic Agent CandidateCompound>

As described above, it has been revealed that the phosphorylations ofthe substrate proteins such as MARCKS are commonly enhanced in themultiple Alzheimer's disease model mice before the onset of the disease.Further, it has also been revealed that the kinase proteins whichphosphorylate these substrate proteins are activated in the Alzheimer'sdisease model mice before the onset of the disease. Thus, based on suchfindings, the present invention makes it possible to provide twoembodiments of a screening method for a candidate compound fordiagnosing Alzheimer's disease described below.

(1) A method comprising the steps of:

bringing a test compound into contact with a phosphorylation site of atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and

selecting the compound if the compound binds to the phosphorylationsite.

(2) A method comprising the steps of:

bringing a test compound into contact with at least one kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF;and

selecting the compound if the compound binds to the kinase protein.

The test compound used in the screening methods of the present inventionis not particularly limited. Examples thereof include expressionproducts of gene libraries, synthetic low-molecular-weight compoundlibraries, peptide libraries, antibodies, substances released frombacteria; liquid extracts and culture supernants of cells(microorganisms, plant cells, animal cells), purified or partiallypurified polypeptides, extracts derived from marine organisms, plants,or animals, soils, and random phage peptide display libraries.

The substrate proteins such as MARCKS and the kinase proteins such asPKC used in these screening methods are as described above in thedescription of <Diagnostic Agent against Alzheimer's Disease>.Nevertheless, the kinase protein is preferably an activated kinaseprotein. Moreover, from the viewpoint of the easiness of the detectionof the binding, a reporter protein (for example, GFP, luciferase), a tagprotein for purification (for example, histidine tag, FLAG tag, GSTtag), or the like may be added to these proteins. Further, theseproteins may be partial peptides, but the substrate proteins and theactivated kinase protein need to contain at least a phosphorylation site(s).

Moreover, the detection of the binding to these proteins is notparticularly limited, and can be performed by selecting a known methodas appropriate. Examples of the known method include aco-immunoprecipitation method, an ELISA method, a method using adetector based on the surface plasmon resonance phenomenon, and a methodbased on FRET (fluorescence resonance energy transfer).

<Alzheimer's Disease Therapeutic Agent 1>

As will be described later in Examples, it has been revealed that thephosphorylations of the substrate proteins such as MARCKS are commonlyenhanced in the multiple Alzheimer's disease model mice before the onsetof the disease. Further, it has also been found that suppressingexpressions of the substrate proteins using shRNA successfullysuppresses a pathology (abnormal spine formation) in the Alzheimer'sdisease model mice.

Thus, the present invention provides an agent for treating Alzheimer'sdisease, the agent comprising a compound capable of suppressing aphosphorylation of at least one substrate protein selected from thegroup consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB(hereinafter, the agent for treating Alzheimer's disease may also bereferred to as simply “therapeutic agent”).

The substrate proteins such as MARCKS targeted by the therapeutic agentof the present invention are as described above in the description of<Diagnostic Agent against Alzheimer's Disease>. In addition,“suppressing” of a phosphorylation of a substrate protein and relatedterms mean to include not only complete suppression (inhibition) butalso partial suppression of the phosphorylation.

Moreover, the suppression of the phosphorylation of the substrateprotein can also be achieved by suppressing the expression of thesubstrate protein per se. Thus, the “compound capable of suppressing thephosphorylation of the substrate protein” also includes a “compoundcapable of suppressing the expression of the substrate protein.”

The “compound capable of suppressing the phosphorylation of thesubstrate protein” is not particularly limited, and may be a knowncompound or may be one identified by the screening to be describedlater. Examples of such a compound include antibodies capable of bindingto a phosphorylated site of the substrate protein, low-molecular-weightcompounds capable of binding to a phosphorylated site of the substrateprotein, RNAs capable of binding to a transcription product of a geneencoding the substrate protein, and peptides having a dominant negativephenotype against the substrate protein. Regarding the antibodies, see<Diagnostic Agent against Alzheimer's Disease>. Regarding such “RNAscapable of binding to a transcription product of a gene encoding aprotein” and “peptides having a dominant negative phenotype against aprotein,” see the description to be described later.

Note that, in the present invention, the term “phosphorylated site”means a site having at least 3 amino acids including one amino acidbefore and one amino acid after a phosphorylated amino acid in a proteinobtained as a result of phosphorylation such as the substrate protein.

<Alzheimer's Disease Therapeutic Agent 2>

As will be described later in Examples, it has also been revealed thatthe kinase proteins which phosphorylate the substrate proteins areactivated in the Alzheimer's disease model mice before the onset of thedisease. Further, it has also been found that suppressing theactivations of the kinase proteins by using an inhibitor against theproteins successfully suppresses the pathology in the Alzheimer'sdisease model mice.

Thus, the present invention also provides, as a second embodiment of thetherapeutic agent against Alzheimer's disease, an agent comprising acompound capable of suppressing an activity or expression of at leastone kinase protein selected from the group consisting of PKC, CaMK, CSK,Lyn, and b-RAF.

The kinase proteins such as PKC targeted by the therapeutic agent of thepresent invention are as described above in the description of<Diagnostic Agent against Alzheimer's Disease>. In addition,“suppressing” of an activity or expression of a kinase protein andrelated terms mean to include not only complete suppression (inhibition)but also partial suppression of the activity or expression.

The “compound capable of suppressing the expression or activity of thekinase protein” is not particularly limited, and may be a known compoundor may be one identified by the screening to be described later.Examples of such a compound include low-molecular-weight compoundscapable of binding to the kinase protein, RNAs capable of binding to atranscription product of a gene encoding the kinase protein, antibodiesagainst the kinase protein, and peptides having a dominant negativephenotype against the kinase protein.

Examples of the low-molecular-weight compound for PKC include PKCinhibitors such as Go6976, UCN-01, BAY43-9006, RO318220, RO320432,Isis3521, LY333531, LY379196, bisindolylmaleimide, sphingosine,staurosporine, midostaurin, tyrphostin 51, hypericin, enzastaurin,rottlerin, safingol, bryostatin 1, perifosine, and ilmofosine. Examplesthereof for CaMK include CaMK inhibitors such as KN-93, KN-62, AIP, CaMkinase II inhibitor 281-301, lavendustin C, K252a, rottlerin, ML-7,ML-9, STO-609, W-7, and W-5. Examples thereof for CSK include CSKinhibitors such as TBCA, IQA, TMCB, quinalizarin, quercetin, andapigenin. Moreover, an example thereof for Lyn includes INNO-406(NS-187). Examples thereof for b-RAF include b-raf inhibitors such asPLX-4720(N-[3-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide),sorafenib(4-[4-[3-[4-chloro-3-(trifluoromethyl)phenyl]ureido]phenoxy]-N-methylpyridine-2-carboxamide),GDC-0879(2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl}ethan-1-ol),vemurafenib (PLX4032, RG7204,N-{3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl}propane-1-sulfonamide),dabrafenib(N-[3-[5-(2-aminopyridin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophenyl]-2,6-difluorobenzenesulfonamide),sorafenib tosylate(4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N²-methylpyridine-2-carboxamidemono(4-methylbenzenesulfonate), and LGX818(methyl[(2S)-1-{[4-(3-{5-chloro-2-fluoro-3-[(methylsulfonyl)amino]phenyl}-1-isopropyl-1H-pyrazol-4-yl)-2-pyrimidinyl]amino}-2-propanyl]carbamate).

In the present invention, examples of the “RNAs capable of binding to atranscription product of a gene encoding a protein” include dsRNAs(double-stranded RNAs), such as siRNAs and shRNAs (short haipin RNAs),complementary to the transcription product of the gene encoding thesubstrate protein or the kinase protein. The length of such a dsRNA isnot particularly limited, as long as the expression of the target genecan be suppressed and no toxicity is demonstrated. The length is, forexample, 15 to 49 base pairs, preferably 15 to 35 base pairs, andfurthermore preferably 21 to 30 base pairs. The dsRNA does notnecessarily have to have completely the same base sequence as that ofthe target gene, but the homology of the sequences is at least 70% ormore, preferably 80% or more, and furthermore preferably 90% or more(for example, 95%, 96%, 97%, 98%, 99% or more). The homology of thesequences can be determined with a BLAST program.

Examples of other forms of the “RNAs capable of binding to atranscription product of a gene encoding a protein” include antisenseRNAs complementary to the transcription product of the gene encoding thesubstrate protein or the kinase protein; and RNAs (ribozymes) having aribozyme activity of specifically cleaving the transcription product.

The above-described RNAs may have some or all of RNAs substituted by anartificial nucleic acid such as PNA, LNA, or ENA. Moreover, in order toexpress these RNAs in a target to which the agent of the presentinvention is administered, each of the RNAs may be in the form of anexpression vector carrying a DNA encoding the RNA. Additionally, thoseskilled in the art can prepare such RNAs by chemical synthesis using acommercially-available synthesizer or the like.

Examples of the “peptides having a dominant negative phenotype against aprotein” for the substrate protein include polypeptides (for example,partial peptides and decoy peptides containing a phosphorylated site ofthe substrate protein) which compete with a kinase protein in binding toa binding site on a substrate protein, and the like. Moreover, examplesthereof for the kinase protein include polypeptides (for example,partial peptides containing a phosphorylated site of the kinase protein)which competitively inhibit the activation of the kinase protein, andthe like.

<Alzheimer's Disease Therapeutic Agent 3>

As described above, it has been revealed that suppressing thephosphorylations of the substrate proteins such as MARCKS successfullysuppresses the pathology in the Alzheimer's disease model mice. Thus,the pathology can also be suppressed by suppressing bindings, which arerequired for the phosphorylations, between the substrate proteins suchas MARCKS and the kinase proteins such as PKC.

Based on such findings, the present invention also provides, as a thirdembodiment of the therapeutic agent against Alzheimer's disease, anagent comprising a compound capable of suppressing a binding of at leastone substrate protein selected from the group consisting of MARCKS,Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH,NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF.

The substrate proteins such as MARCKS and the kinase proteins such asPKC targeted by the therapeutic agent of the present invention are asdescribed above in the description of <Diagnostic Agent againstAlzheimer's Disease>. Nevertheless, the substrate proteins arepreferably not phosphorylated, while the kinase proteins are preferablyactivated kinase proteins. In addition, “suppressing” of a bindingbetween these proteins and related terms mean to include not onlycomplete suppression (inhibition) but also partial suppression of thebinding.

The “compound capable of suppressing the binding” is not particularlylimited, and may be a known compound or may be one identified by thescreening to be described later. Examples of such a compound includepolypeptides, antibodies, and low-molecular-weight compounds all ofwhich compete with the substrate proteins and the kinase proteins inbinding to a binding site on the substrate proteins or the kinaseproteins. Note that the low-molecular-weight compounds of the presentinvention also include physiologically acceptable salt or solvate formsof the low-molecular-weight compounds.

<Alzheimer's Disease Therapeutic Agent 4>

As will be described later in Examples, it has been revealed thatactivating Lyn also successfully suppresses the pathology in theAlzheimer's disease model mice. Thus, the present invention alsoprovides, as a therapeutic agent against Alzheimer's disease, an agentcomprising a compound capable of activating Lyn. Lyn targeted by thetherapeutic agent of the present invention is as described above in thedescription of <Diagnostic Agent against Alzheimer's Disease>.

The “compound capable of activating Lyn” is not particularly limited,and may be a known compound. Examples of such a compound includelow-molecular-weight compounds capable of binding to Lyn. Moreconcretely, examples thereof include Lyn kinase activators described inInternational Publication No. WO2008/103692 (MLR-1023 and the like).

In addition, increasing an amount of Lyn expressed can also increase theactivity of Lyn. Thus, the “compound capable of activating Lyn” alsoincludes: nucleic acids (DNA, RNA) encoding Lyn; DNA constructs (forexample, plasmid DNA, viral vector) capable of expressing Lyn, which isencoded by the nucleic acids, in target cells; and Lyn proteins.

Hereinabove, preferred embodiments of the therapeutic agent of thepresent invention have been described. In addition, the therapeuticagent of the present invention may comprise, besides the above-describedcompounds, the aforementioned other pharmacologically acceptableingredients, as in the case of the diagnostic agents described above.Further, the therapeutic agent of the present invention may alsocomprise a carrier for introducing a nucleic acid, a protein, or thelike into cells. Examples of the carrier include substances having apositive charge such as cationic liposome, and lipophilic substances(cholesterols and derivatives thereof, lipids (such as, for example,glycolipids, phospholipids, sphingolipids), vitamins such as vitamin E(tocopherols)). Additionally, the therapeutic agent of the presentinvention may be used in combination with known pharmaceutical drugswhich are used in the treatment of Alzheimer's disease.

The mode of administering the therapeutic agent of the present inventionis not particularly limited, and examples thereof include intravenousadministration, intraarterial administration, intraperitonealadministration, subcutaneous administration, intradermal administration,tracheobronchial administration, rectal administration and intramuscularadministration, administration by transfusion, and direct administrationinto a target site (such as brain). From the viewpoints that thetherapeutic effect is high and that an amount of the agent to beadministered is small, the direct administration into a target site ispreferable. The administration to a target site can be achieved byemploying, for example, cannula (catheter), surgical incision, drugdelivery system, injection, or the like. More concretely, examplesthereof include a method in which a cannula or the like is inserted bystereotactic surgery to administer the agent into the brain through thecannula; a method in which after a craniotomy, a sustained-release drugdelivery system (for example, ALZET osmotic pump) with the agent isimplanted into the brain; and a method in which the agent is introducedinto cells in the brain by electropolation. Meanwhile, in the case wherethe agent of the present invention is not directly administered into thebrain, it is possible to utilize a method in which a brainbarrier-permeable substance is bound to the compound and administered.Note that an example of the brain barrier-permeable substance includes a29-amino-acid glycoprotein derived from rabies virus (see Nature, 2007Jul. 5, Vol. 448, pp. 39 to 43), but is not limited thereto.

An amount of the therapeutic agent of the present invention administeredand the number of administrations can be adjusted as appropriatedepending on the type of the compounds, the body weight and symptom of atest subject, and the like. The number of administrations can beadjusted as appropriate depending on the administration amount, theadministration route, and the like.

A product of the therapeutic agent of the present invention or a manualthereof may be provided with an indication stating that the product isused for treating Alzheimer's disease. “A product or a manual providedwith an indication” is as described above in the description of<Diagnostic Agent against Alzheimer's Disease>. The indication mayinclude information on an action mechanism of the agent of the presentinvention such as information that administering the agent of thepresent invention suppresses phosphorylations of the substrate proteinssuch as MARCKS, thereby suppressing abnormal spine formation or thelike, and alleviating a pathology of Alzheimer's disease.

Moreover, the present invention also makes it possible to treatAlzheimer's disease by administering the compound to a subject asdescribed above. Thus, the present invention also provides a method fortreating Alzheimer's disease, the method characteristized by comprisingadministering to a subject any one of: a compound capable of suppressinga phosphorylation of at least one substrate protein selected from thegroup consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; acompound capable of suppressing an activity or expression of at leastone kinase protein selected from the group consisting of PKC, CaMK, CSK,Lyn, and b-RAF; a compound capable of suppressing a binding of at leastone substrate protein selected from the group consisting of MARCKS,Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH,NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF;and a compound capable of activating Lyn.

<Screening Method 1 for Alzheimer's Disease Therapeutic Agent CandidateCompound>

As described above, it has been found that suppressing the expressionsof the substrate proteins such as MARCKS in the Alzheimer's diseasemodel mice suppresses the phosphorylations of the protein, therebysuccessfully suppressing the pathology in the mice. Thus, based on suchfindings, the present invention also provides the following screeningmethod for a candidate compound for treating Alzheimer's disease, themethod comprising:

(i) a step of applying a test compound to a system capable of detectinga phosphorylation of at least one substrate protein selected from thegroup consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and

(ii) a step of selecting the compound if the compound suppresses thephosphorylation of the substrate protein.

The “substrate proteins such as MARCKS” used in this screening methodare as described above in the description of <Screening Method forAlzheimer's Disease Diagnostic Agent Candidate Compound>. These proteinsmay be partial peptides, but need to contain at least a phosphorylatedsite(s). Moreover, other proteins such as a tag protein for purificationmay be added to these proteins.

Further, the “test compound” used in this screening method is notparticularly limited. Examples thereof include the compounds describedabove in <Screening Method for Alzheimer's Disease Diagnostic AgentCandidate Compound>.

The “system capable of detecting the phosphorylation of the substrateprotein” is not particularly limited. An example thereof includes amixture solution of the substrate proteins such as MARCKS and the kinaseproteins (such as PKC) which phosphorylate the substrate proteins.Moreover, a radiolabeled phosphate is added together with a testcompound to this mixture solution, followed by incubation to detect theincorporation of the phosphate into the substrate protein with ascintillation counter, by autoradiography, or other means. Then, if anamount of the phosphate incorporated into the substrate protein detectedin the presence of the test compound is small in comparison with thatdetected in the absence of the test compound, the test compound isevaluated as a compound which suppresses the phosphorylation of thesubstrate protein, and selected as a candidate compound for treatingAlzheimer's disease.

Additionally, an example of another embodiment of the “system capable ofdetecting the phosphorylation of the substrate protein” includes asystem capable of directly detecting the phosphorylation of thesubstrate protein such as MARCKS. Regarding this system, a test compoundis applied to cells expressing the substrate protein or cells in whichthe protein is forcibly expressed, and the phosphorylation of thesubstrate protein in the cells is detected. Then, if an amount of thephosphorylated substrate protein detected is small in comparison withthat detected in the absence of the test compound, the test compound isevaluated as a compound which suppresses the phosphorylation of thesubstrate protein. In the case where the phosphorylation of thesubstrate protein is directly detected, it is suitable to employ adetection method using an antibody capable of specifically binding to aphosphorylation site of a substrate protein, a method utilizing adetector based on the surface plasmon resonance phenomenon using a thinmetal film on which a compound capable of specifically binding to aphosphorylation site of a substrate protein is immobilized, and the likeas in <Method 1 for Diagnosing Alzheimer's Disease> described above.

<Screening Method 2 for Alzheimer's Disease Therapeutic Agent CandidateCompound>

As described above, it has been found that suppressing activations ofthe kinase proteins such as PKC in the Alzheimer's disease model micealso successfully suppresses the pathology in the mice. Thus, based onsuch a finding, the present invention also provides the following methodas a second embodiment of the screening method for a candidate compoundfor treating Alzheimer's disease, the method comprising:

(i) a step of applying a test compound to a system capable of detectingan activity or expression of at least one kinase protein selected fromthe group consisting of PKC, CaMK, CSK, Lyn, and b-RAF; and

(ii) a step of selecting the compound if the compound suppresses theactivity or expression of the protein.

As in <Method for Diagnosing Alzheimer's Disease> described above, the“kinase proteins such as PKC” used in this screening method are notlimited respectively to the proteins having the amino acid sequenceslisted as the typical examples described above, and naturally-occurringmutants thereof can also be targeted.

The “system capable of detecting the activity of the kinase protein suchas PKC” should be a system capable of detecting the activity of thekinase protein, that is, the phosphorylation of the substrate protein.It is suitable to use the systems described above in <Screening Method 1for Alzheimer's Disease Therapeutic Agent Candidate Compound>. Moreover,since a phosphorylation of a substrate protein requires the activation(such as phosphorylation) of a kinase protein per se, a “system capableof detecting a phosphorylation of a kinase protein such as PKC” may alsobe used. Note that this system is constructed using the kinase proteinssuch as PKC in place of the substrate proteins such as MARCKS in the“system capable of detecting the phosphorylation of the substrateprotein” described above.

Examples of the “system capable of detecting the expression of thekinase protein such as PKC” include cells having a DNA in which areporter gene is operably linked downstream of a promoter region of agene encoding the each kinase protein, or liquid extracts from thecells. Herein, the phrase “operably linked” refers to linking of thereporter gene to the promoter region of each gene such that theexpression of the reporter gene is induced by binding of a transcriptionfactor to the promoter region of the gene. Moreover, a test compound isapplied to this system to measure an activity of a protein encoded bythe reporter gene. If the detected activity is low in comparison withthat detected in the absence of the test compound, the test compound isevaluated as having an activity of suppressing the expression of eachkinase protein.

An example of another embodiment of the “system capable of detecting theexpression of the kinase protein such as PKC” besides theabove-described reporter system includes a system capable of directlydetecting the expression of the kinase protein such as PKC. Regardingthis system, a test compound is applied to cells expressing eachprotein, and the expression of each protein in the cells is detected.Then, if an detected amount of each protein expressed is small incomparison with that detected in the absence of the test compound, thetest compound is evaluated as having an activity of suppressing theexpression of the protein. In detecting the expression of the protein,in the case where the expression of the protein per se is to bedetected, it is suitable to employ a detection method using an antibodycapable of specifically binding to a kinase protein (preferably,activated kinase protein), a method utilizing a detector based on thesurface plasmon resonance phenomenon using a thin metal film on which acompound capable of specifically binding to a kinase protein(preferably, activated kinase protein) is immobilized, and the like asin <Method 2 for Diagnosing Alzheimer's Disease> described above.Meanwhile, in a case of detecting the expression of the kinase proteinthrough the gene expression at a transcription level, a northernblotting method, an RT-PCR method, a dot blotting method, or the likecan be employed.

<Screening Method 3 for Alzheimer's Disease Therapeutic Agent CandidateCompound>

As described above, it has been revealed that suppressing thephosphorylations of the substrate proteins such as MARCKS successfullysuppresses the pathology in the Alzheimer's disease model mice. Thus,the pathology can also be suppressed by suppressing bindings, which arerequired for the phosphorylations, between the substrate proteins suchas MARCKS and the kinase proteins such as PKC.

Based on such findings, the present invention also provides thefollowing method as a third embodiment of the screening method for acandidate compound for treating Alzheimer's disease.

A screening method for a candidate compound for treating Alzheimer'sdisease, the method comprising the following steps (a) to (c):

(a) a step of bringing at least one kinase protein selected from thegroup consisting of PKC, CaMK, CSK, Lyn, and b-RAF into contact with atleast one substrate protein selected from the group consisting ofMARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A,CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB, in presence of a testcompound;

(b) a step of detecting a binding between the kinase protein and thesubstrate protein; and

(c) a step of selecting the compound if the compound suppresses thebinding.

The “kinase proteins such as PKC,” the “substrate proteins such asMARCKS,” and the “test compound” used in this screening method are asdescribed above in the description of <Screening Method for Alzheimer'sDisease Diagnostic Agent Candidate Compound>. Nevertheless, thesubstrate proteins are preferably not phosphorylated, while the kinaseproteins are preferably activated kinase proteins.

In the step (a), the kinase protein and the substrate protein arebrought into contact with each other in the presence of the testcompound. The contact should be conducted under conditions that wouldnot inhibit the binding in the absence of the test compound.

In the step (b), the binding between the kinase protein and thesubstrate protein is detected. This binding detection is notparticularly limited, and a known method can be employed as appropriate.For example, it is possible to employ a co-immunoprecipitation method,an ELISA method, a method using a detector based on the surface plasmonresonance phenomenon, or a method based on FRET.

In the step (c), the compound is selected if the compound suppresses thebinding. For example, when the co-immunoprecipitation method isemployed, the evaluation is possible by comparison between an amount ofthe substrate protein coprecipirated when the kinase protein isprecipitated by an antibody specific thereto in the presence of the testcompound and an amount (control value) of the substrate protein in theabsence of the test compound. To be more specific, if the amount of thesubstrate protein in the presence of the test compound is small incomparison with the amount in the absence of the test compound, the testcompound can be evaluated as a candidate compound for treatingAlzheimer's disease. When a method other than the immunoprecipitationmethod is employed in the detection of the binding, a similar evaluationis possible by using the degree of the binding in the absence of thetest compound as a control value.

Hereinabove, preferred embodiments of the screening method for acandidate compound for treating Alzheimer's disease of the presentinvention have been described. In addition, in the screening method ofthe present invention, it is possible to further narrow down a candidatecompound on the basis of a recovery from a symptom of an Alzheimer'sdisease model animal in which the compound selected according to theabove-described methods has been administered.

Examples of the “Alzheimer's disease model animals” include, asdescribed later in Examples, animals (such as mice, rats, marmosets) inwhich an Alzheimer's disease responsible gene (such as PS1 exon 9deletion mutant; PS2 mutant (N141I); human double-mutant APP695(KM670/671NL, Swedish type); quintuple mutant (human APP695 triplemutant with Swedish type (KM670/671NL), Florida type (I716V), and Londontype (V717I), as well as human PS1 double mutant (M146L and L285V));human tau mutant; or a combination of these mutants) is introduced.

The “recovery from a symptom of a model animal” can be detected, forexample, as described later in Examples, by performing in vivo imagingwith a two-photon microscope on the degree of a recovery from abnormalspine formation caused by Alzheimer's disease. Moreover, the detectionis also possible by conducting a behavioral test described later inExamples and evaluating the degree of a recovery from an abnormalbehavior of the model animal.

Hereinabove, preferred embodiments of the diagnosis method, thediagnostic agent, and the therapeutic agent against Alzheimer's disease,the screening methods for candidate compounds of these agents of thepresent invention, and so forth have been described. Hereinafter,description will be given of a diagnostic agent, a therapeutic agent,and so forth against frontotemporal lobar degeneration.

<Method for Diagnosing Frontotemporal Lobar Degeneration>

As will be described later in Examples, it has been revealed that ab-RAF protein which is a kinase protein phosphorylating substrateproteins such as tau protein is activated in frontotemporal lobardegeneration model mice before the onset of the disease. Thus, thepresent invention also provides a method for diagnosing frontotemporallobar degeneration, the method comprising the following the steps (i) to(iii):

(i) a step of detecting an activity or expression of a b-RAF protein ina test subject;

(ii) a step of comparing the activity or expression with an activity orexpression of a b-RAF protein in a normal subject; and

(iii) a step of determining that the test subject is affected withfrontotemporal lobar degeneration or has a risk of developingfrontotemporal lobar degeneration if the activity or expression of theb-RAF protein in the test subject is higher than the activity orexpression of the b-RAF protein in the normal subject as a result of thecomparison.

This diagnosis method is a method similar to <Method for DiagnosingAlzheimer's Disease> described above. In addition, examples of the“activated kinase protein” to be detected include, as described above,b-RAF having serine at position 365 phosphorylated, b-RAF having serineat position 446 phosphorylated, b-RAF having serine at position 579phosphorylated, b-RAF having threonine at position 599 phosphorylated,b-RAF having serine at position 602 phosphorylated, b-RAF having serineat position 729 phosphorylated, and b-RAF having serine at position 732phosphorylated. From the viewpoint of having a larger difference betweena subject affected with frontotemporal lobar degeneration or having arisk of developing and a normal subject, the detection target in themethod for diagnosing frontotemporal lobar degeneration of the presentinvention is preferably b-RAF having serine at position 365phosphorylated, b-RAF having serine at position 729 phosphorylated, andb-RAF having serine at position 732 phosphorylated, and the detectiontarget is more preferably b-RAF having serine at position 729phosphorylated.

Moreover, the present invention makes it possible to determine that oneis affected with frontotemporal lobar degeneration or has a risk ofdeveloping frontotemporal lobar degeneration. In this manner, enablingjudgment of frontotemporal lobar degeneration affection or the like atan early stage leads to an expectation that treatment methods forsuppressing a pathology of frontotemporal lobar degeneration (such amethod for administering an agent for suppressing a pathology offrontotemporal lobar degeneration) will be effective.

Thus, the present invention also makes it possible to provide, as in thecase of <Method for Diagnosing Alzheimer's Disease> described above, amethod for treating frontotemporal lobar degeneration, the methodcomprising a step of administering an agent for suppressing a pathologyof frontotemporal lobar degeneration to a test subject determined to beaffected with frontotemporal lobar degeneration or have a risk ofdeveloping frontotemporal lobar degeneration by the diagnosis method ofthe present invention.

Additionally, examples of the agent administered to suppress a pathologyof frontotemporal lobar degeneration include serotonin-specific reuptakeinhibitors (SSRI), cholinesterase inhibitors (ChEI), agents forsuppressing tau aggregation (such as aminothienopyridazine, cyanine dye,methylene blue), and neuroprotective drugs (such as dimebon).

Further, as will be described later, it is also possible to suitablyuse, as the agent for suppressing a pathology of frontotemporal lobardegeneration, an agent for treating frontotemporal lobar degeneration,the agent comprising a compound capable of suppressing an activity orexpression of a b-RAF protein.

<Diagnostic Agent Against Frontotemporal Lobar Degeneration>

As described above, it has been revealed that the kinase protein b-RAFwhich phosphorylates the substrate proteins such as tau protein isactivated before the onset of the disease. Thus, the present inventionprovides an agent for diagnosing frontotemporal lobar degeneration, theagent comprising a compound having an activity of binding to b-RAF.

As in the case of <Method for Diagnosing Alzheimer's Disease> describedabove, the b-RAF protein targeted by the diagnostic agent againstfrontotemporal lobar degeneration of the present invention is notlimited to the proteins having the amino acid sequences listed as thetypical examples described above, and naturally-occurring mutants canalso be targeted. Moreover, the “compound having an activity of bindingto b-RAF” is not particularly limited, and it is possible to similarlyuse the compounds described above in <Diagnostic Agent againstAlzheimer's Disease>.

<Therapeutic Agent Against Frontotemporal Lobar Degeneration>

As will be described later in Examples, it has also been revealed thatthe b-RAF protein which phosphorylates the substrate proteins such astau protein is activated in the frontotemporal lobar degeneration modelmice before the onset of the disease. Further, it has also been foundthat suppressing the activation of the b-RAF protein by using aninhibitor against the protein successfully suppresses the pathology inthe frontotemporal lobar degeneration model mice.

Thus, the present invention also provides, as a therapeutic agentagainst frontotemporal lobar degeneration, an agent comprising acompound capable of suppressing an activity or expression of b-RAF.

The frontotemporal lobar degeneration therapeutic agent of the presentinvention is similar to <Alzheimer's disease therapeutic agent>described above. Moreover, the targeted b-RAF protein is as describedabove in the description of <Diagnostic Agent against FrontotemporalLobar Degeneration>.

As in the case of <Alzheimer's disease therapeutic agent> describedabove, a product of the frontotemporal lobar degeneration therapeuticagent of the present invention or a manual thereof may be provided withan indication stating that the therapeutic agent is used for treatingfrontotemporal lobar degeneration. “A product or a manual provided withan indication” is as described above in the description of <DiagnosticAgent against Alzheimer's Disease>. The indication may includeinformation on an action mechanism of the agent of the present inventionsuch as information that administering the agent of the presentinvention suppresses b-RAF activity, thereby suppressing a decrease inthe number of spines and so forth, and alleviating a pathology offrontotemporal lobar degeneration.

Moreover, the present invention also makes it possible to treatfrontotemporal lobar degeneration by administering the compound to asubject as described above. Thus, the present invention also provides amethod for treating frontotemporal lobar degeneration, the methodcharacterized by comprising administering to a subject a compoundcapable of suppressing an activity or expression of b-RAF.

EXAMPLES

Hereinafter, the present invention will be more specifically describedon the basis of Examples. However, the present invention is not limitedto the following Examples.

—Alzheimer's Disease—

In the present Examples, first, experimental methods and so forthdescribed below were carried out to identify phosphoproteins and kinaseproteins which played central roles in a pre-onset stage of Alzheimer'sdisease, as well as a network composed of these proteins, andconsequently to provide target molecules useful in the diagnosis andtreatment of Alzheimer's disease.

<Experiments Using Model Mice>

The following five types of Alzheimer's disease model mice were used inthe present Examples.

(1) PS1 transgenic mice (mice expressing exon 9 deletion mutant(PSEN1dE9) under the control of the mouse PrP promoter; see Jankowsky,J. L. et al., Hum. Mol. Genet., 2004, Vol. 13, pp. 159 to 170)(2) PS2 transgenic mice (mice expressing human PS2 mutant (N141I) underthe control of the ubiquitous CMV early enhancer and the chicken R actinpromoter; see Oyama, F. et al., J. Neurochem., 1998, Vol. 71, pp. 313 to322)(3) Human double-mutant APP695 (KM670/671NL, Swedish type) transgenicmice (the mice were prepared by substituting a mutant for the PrP genein a hamster PrP cosmid vector (see Hsiao, K. et al., Science, 1996,Vol. 274, pp. 99 to 102))(4) 5×FAD mice (transgenic mice expressing human APP695 having Swedishtype (KM670/671NL), Florida type (I716V), and London type (V717I) triplemutations, as well as human PS1 having double mutations (M146L andL285V) under the control of mouse Thy1) (see Oakley, H. et al., J.Neurosci., 2006, Vol. 26, pp. 10129 to 10140)(5) Transgenic mice expressing a human tau mutant protein under thecontrol of the mouse PrP promoter (see Yoshiyama, Y. et al., Neuron,2007, Vol. 53, pp. 337 to 351).

Note that the genetic backgrounds of the transgenic mice were C57BL/6J,C57BL/6J, C57/B6XSJL, C57/B6XSJL, and B6C3H/F1, respectively.

In the mass spectrometry to be described later, brain tissues wereisolated from male transgenic mice described above at the age in monthsshown in figures and descriptions thereof, and subjected to theanalysis.

In the immunohistochemical analysis, the brain samples were fixed with4% paraformaldehyde, and paraffin sections were prepared (the thicknessof each section: 5 μm) using a microtome (manufactured by Yamato KohkiIndustrial Co., Ltd.). Meanwhile, the following antibodies were eachdiluted to 1/1000 and used as primary antibodies.

Anti-Aβ antibody (82E1), manufactured by IBL Co., Ltd., Code No: 10323Anti-Aβ antibody (6E10), manufactured by Covance Inc., Product Code:SIG-39300Anti-human PHF-tau antibody (AT-8), manufactured by Innogenetics N.V.,Catalog No: BR-03.

Then, the tissue samples reacted with each antibody were treated withVECTASTAIN Elite ABC Kit and DAB Peroxidase Substrate Kit (manufacturedby Vector Laboratories) to visualize the expressions of proteinsrecognized by the antibodies.

Moreover, although unillustrated, male transgenic mice described abovewere subjected to six behavioral tests described below. Based ondetected abnormal behaviors, whether or not these mice developedAlzheimer's disease was evaluated.

(1) Morris Water Maze Test

In this test, the mice received a 60-second trial four times a day for 5days. The time until each mouse reached the platform was measured.

(2) Rotarod Test

In this test, a trial was conducted four times a day for 3 days in whicha mouse was allowed to grab on a rotating rod (rotation speed: 3.5 to 35rpm) with the speed being gradually increased. The average time untilthe mouse fell from the rotating rod was recorded.

(3) Fear-Conditioning Test

In this test, first, a mouse received a sound stimulus (65-dB whitenoise, 30 seconds) together with an electrical stimulus (0.4 mA, 2seconds) on the foot. Then, after 24 hours, the mouse was measured forthe frequency of the freezing reaction when the mouse received a soundstimulus but no electrical stimulus in the same chamber.

(4) Open-Field Test

In this test, the time during which a mouse stayed in a central regionof an open field was measured.

(5) Light-Dark Box Test

In this test, the time during which a mouse stayed in a light box wasmeasured.

(6) Elevated Plus Maze Test

In this test, the time during which a mouse stayed on arms with no wallsin an elevated plus maze set 60 cm above the floor was measured.

<Human Brain>

For a proteome analysis to be described later, brain samples wereisolated from AD (Alzheimer's disease) patients, DLB (dementia with lewybodies) patient, and healthy control persons and frozen at −80° C.within 1 hour after death. Moreover, temporal pole and occipital poletissues were dissected from five brains in each group.

Note that a neuropathologist pathologically diagnosed each brain samplebased on the immunohistochemistry. As a result, in the brains of the ADpatients, other pathologies such as lewy bodies, TDP43 cytoplasmicaggregates, and argyrophilic grains were not observed. Moreover, in thebrains of the DLB patients, the disease-specific pathologicalobservation was confirmed.

<Preparation of Phosphoproteins and Phosphopeptides>

In preparing phosphoproteins and phosphopeptides from the transgenicmice and so forth, first, mice were euthanized using ethyle ether.Within 5 minutes thereafter, the cerebral cortexes were collected. Theobtained cerebral cortexes were immediately frozen with liquid nitrogenand stored until phosphoproteins were extracted. In the proteinextraction, first, the cortical tissues were lysed with a cold lysisbuffer containing 2% SDS, 1 mM DTT, and 100 mM Tris-HCl (pH 7.5). Thecells were disrupted with 20 strokes of a glass Dounce homogenizer onice. The ratio of the lysis buffer to the tissue was 10 μL to 1 mg.After the cells were disrupted, the lysate was incubated at 100° C. for15 minutes. Then, the crude extract was obtained by centrifugation at 4°C. at 16000×g for 10 minutes. The collected supernant was diluted to a1/10 concentration with water, and filtered through a filter having apore diameter of 0.22 μm. The resulting flow-through fraction wasconcentrated to a 10-fold concentration using an Amicon Ultra 3K filter(manufactured by Millipore Corporation). Further, the concentrations ofproteins thus prepared were measured using the BCA Protein Assay Reagent(manufactured by Thermo Fisher Scientific Inc.).

Subsequently, a solution of 100 μL of 1 M triethylammonium bicarbonate(TEAB) (pH 8.5), 3 μL of 10% SDS, and 30 μL of 50 mM tris-2-carboxyethylphosphine (TCEP) was added to sample aliquots (200 μL) containing 15 mgof the proteins, and incubated at 60° C. for 1 hour. Moreover, toprotect cysteine residues, 10 mM methyl methanethiosulfonate (MMTS) wasadded and treated at 25° C. for 10 minutes. Thereafter, the obtainedsample was treated at 37° C. for 24 hours with 80 mM CaCl₂ and trypsin(mass spectrometry grade) (10:1 protein/enzyme, w/w). Then,phosphopeptides were concentrated using TITANSPHERE(registeredtrademark) Phos-Tio Kit (manufactured by GL Sciences Inc.) according tothe instruction, and desalted using a Sep-Pak Light C18 cartridge column(manufactured by Waters Corporation) according to the instruction. Thesample aliquots were dried and then dissolved in 25 μL of 100 mM TEAB(pH 8.5). Further, the phosphopeptide in each sample were labeledseparately using the iTRAQ(registered trademark) multiplex assay kit(manufactured by AB SCIEX Ins.) at 25° C. for 2 hours according to theinstruction. Subsequently, the labeled phosphopeptide pools were mixedtogether. The obtained aliquots were dried and then re-dissolved in 1 mLof 0.1% formic acid.

<2D LC MS/MS Analysis>

The phosphopeptide samples labeled as described above were subjected tostrong cation exchange (SCX) chromatography using a TSK gel SP-5PWcolumn (manufactured by TOSHO Corporation) and a Prominence UFLC system(manufactured by Shimadzu Corporation). Note that the flow rate was 10mL/minute with solution A (10 mM KH₂PO₄ (pH 3.0), 25% acetonitrile).Thereafter, elution was performed using solution B (10 mM KH₂PO₄ (pH3.0), 25% acetonitrile, 1 M KCl) in a gradient range of 0 to 50%. Thecollected elution fractions were dried and then re-dissolved in 100 μLof 0.1% formic acid.

Subsequently, each fraction thus prepared was analyzed using a DiNaNano-Flow LC system (manufactured by KYA Technologies Corporation) andTriple TOF 5600 System (manufactured by AB SCIEX Ins.). In the liquidchromatography, samples were loaded onto a 0.1 mm×100 mm C18 columntogether with solution C (2% acetonitrile and 0.1% formic acid) andeluted using solution D (80% acetonitrile and 0.1% formic acid) in agradient range of 0 to 50%. Note that the flow rate was set at 300nL/minute, and the ion spray voltage was set at 2.3 kV. Theinformation-dependent acquisition (IDA) was performed in a range of 400to 1250 m/z with 2 to 5 charges. Moreover, to identify each peptide, theAnalyst TF1.5 software (manufactured by AB SCIEX Ins.) was used.Further, each peptide was quantified based on the TOF-MS currentdetected during the LC-separated peptide peak, and adjusted to thecharge/peptide ratio. In addition, the obtained signals were analyzedusing Analyst TF (version 1.5) (manufactured by AB SCIEX Ins.). Then,the signals were processed by ProteinPilot software (version 4).

<Data Analysis>

As described above, in the 2D LC MS/MS analysis, the mass spectra of thepeptides were acquired and analyzed using Analyst TF (version 1.5)(manufactured by AB SCIEX Ins.). Then, based on the obtained result,corresponding proteins were searched using human and mouse proteinsequence database (UniProtKB/Swiss-Prot, data downloaded from UniProt(www.uniprot.org) on 2010 Jun. 22, with ProteinPilot software (version4) including Paragon algorithm (manufactured by AB SCIEX Ins., seeShilov, I. V. et al., Mol. Cell. Proteomics, 2007, Vol. 6, pp. 1638 to1655) as described above. Note that the tolerance for the searched ofthe peptides by ProteinPilot was set to 0.05 Da for the MS analysis and0.10 Da for the MS/MS analysis. Moreover, in ProteinPilot,“phosphorylation emphasis” was set at the sample description, and“biological modifications” was set at the processing specification.Further, the confidence score was utilized to evaluate the quality ofthe peptide identification. Furthermore, the identified proteins weregrouped by the ProGroup algorithm (manufactured by AB SCIEX Ins.) toexclude redundancy. Additionally, the threshold value for the proteindetection was set at 95% confidence in ProteinPilot. Then, if theconfidence was 95% or more, the protein was determined to be identified.

Moreover, an MS/MS spectrum was prepared upon a fragmentation in themass spectrometer. Further, the proteins were quantified through iTRAQreporter group analysis in the MS/MS spectrum. In the peptide andprotein quantification, bias correction option was used to normalizesignals of different iTRAQ reporters. In addition, peptide ratios, thatis, ratios between reporter signals in the AD patients and those incontrol samples, were calculated after the bias correction. A proteinratio (average ratio) was deduced from a weighted average of peptideratios corresponding to proteins. Moreover, the deduction used peptideratios differently weighted based on error factors after the biascorrection. Note that detailed formulas used to calculate these valueswere described in the manual from ABSCIEX. Further, using the peptideratios, amounts of the proteins in the AD patients were compared withthose in the control samples. Student's t-value was calculated fromweighted average of log peptide ratio, its standard error, and log bias.Furthermore, P-value was calculated together with a post hoc test inProteinPilot to exclude multiple hypothesis testing-related problem. TheP-values of three samples obtained in this test were integrated byinverse normal method. Then, if the integrated P-value was smaller than0.05, it was determined that the phosphorylations of the proteins werechanged.

The peptide summary and protein summary in ProteinPilot were inputtedinto Excel for further data analyses. Moreover, a geometric mean ofsignal intensities derived from multiple MS/MS fragments containing thephosphorylation site was calculated as an amount of phosphopeptidefragment. Further, a difference between the AD patient group and thecontrol group was evaluated by Student's t-test (n=3). Then, the changedphosphoproteins were compared among different AD models, and proteinswere selected which commonly changed in a hypothesis free approach or anAβ aggregation-linked approach.

<Systems Biology>

ProteinPilot software was used to identify proteins expressed in theoccipital lobe and the temporal lobe of the human brain (see Shilov, I.V. et al., Mol. Cell. Proteomics, 2007, Vol. 6, pp. 1638 to 1655). To bemore specific, ProteinPilot automatically added Uniprot ID to eachobserved protein. Then, the observed proteins were searched for proteinsbelonging to common Homologene Group ID, and the Taxonomy IDs and GeneIDs of the collected proteins were obtained. Note that the number of theTaxonomy IDs was limited to 9606 for human, 10090 for mouse, and 10116for rat. Moreover, Uniprot IDs of newly added proteins were alsoattached. Next, from the list of the collected proteins, proteins havingUniprot IDs not listed in the GNP database(http://genomenetwork.nig.ac.jp/index_e.html) were excluded.Subsequently, a database was created from information collected from theGNP by utilizing a super computer system at the Human Genome Center inthe University of Tokyo. As a result, remaining proteins were determinedas analyzed proteins. Moreover, the GNP database was searched forproteins linked to the analyzed proteins, so that an edge file wascreated (redundant edges were excluded). Based on the created edge file,a protein network was obtained and visualized using Cell Illustrator(see Nagasaki, M. et al., Appl. Bioinformatics, 2003, Vol. 2, pp. 181 to184).

<In Vivo Imaging with Two-Photon Microscope>

Two-photon imaging of dendritic spine was performed using alaser-scanning microscope system FV1000MPE2 (manufactured by OlympusCorporation) equipped with an upright microscope (BX61WI, manufacturedby Olympus Corporation, a water-immersion objective lens (XLPlanN25xW;numerical aperture, 1.05), and a pulsed laser (MaiTai HP DeepSee,manufactured by Spectra Physics). In the imaging, EGFP was excited bylight at a wavelength of 890 nm, and scanned in a range of 500 to 550nm. Moreover, the scanned region for three-dimensional imaging was100×100 μm (1 μm Z-axis steps, 1024×1024 pixels).

Additionally, two weeks before the imaging, adeno-associated virus 1(AAV1)-EGFP with the synapsin 1 promoter (titer: 1×10¹⁰ vectorgenomes/mL, 1 μL) was injected into the retrosplenial cortex (−2.0 mmanteroposterior and 0.6 mm mediolateral from the bregma, depth 1 mm) ofmice under anesthesia with 2.5% isoflurane. Then, two weeks thereafter,the dendritic spines of the first layer (layer 1) of the cerebral cortexwere observed through a thinned skull window according to the methoddescribed in “Yang, G. et al., Nat. Protoc., 2010, Vol. 5, pp. 201 to208.”

Moreover, when the influence of kinase inhibition on dendritic spineswas imaged, Alzet micro-osmotic pumps (model: 1003D, manufactured byDurect Corporation) filled with PBS/1% DMSO containing 1 μM Go6976(manufactured by Calbiochem), 0.4 mM KN-93 (manufactured by CaymanChemical), or 1 μM MLR1023 (manufactured by Glixx Laboratories) wereimplanted into mice under anesthesia with O₂/isoflurane. Then, 30 hoursor 60 hours elapsed after the osmotic pumps were implanted, dendriticspines were observed. Note that, regarding the PKC inhibitor Go6976, seeYan, Z. et al., Proc. Natl. Acad. Sci. U.S.A., 1999, Vol. 96, pp. 11607to 11612. Regarding the CaMK inhibitor KN-93, see Galan, A. et al.,Pain, 2004, Vol. 112, pp. 315 to 323. Regarding the Lyn activatorMLR1023, see Saporito, M. S. et al., J. Pharmacol. Exp. Ther., 2012,Vol. 342, pp. 15 to 22.

Meanwhile, when the influence of shRNA-lentiviral vector introduction ondendritic spines was imaged, 3 μL of a lentiviral vector encoding shRNAagainst MARCKS (sc-35858-V, manufactured by Santa Cruz BiotechnologyInc., 1×10⁶ TU) or scrambled shRNA (RHS4348, 1×10⁶ TU) was injected intothe same region as in the case of the AAV1-EGFP.

In addition, the spine density, spine length, spine maximum diameter,and spine neck minimum diameter were measured from the obtained imagesusing image analysis software IMARIS 7.2.2 (manufactured by Bitplane).

<Statistical Analysis>

Mass spectrometry data on the disease model mice or human patients wereevaluated by inverse normal method in comparison with data on therespective background mice or human control samples. The amount of eachpeptide in the mass spectrometry was based on multiple peaks, and theamount of each protein was based on multiple peptide values. Inconsideration of these, P-values were obtained for these amounts.Moreover, together with the P-values, differential gene expressionanalysis was performed on each peptide or protein without replication.Further, to guarantee the result quality, biologically replicated datawere also obtained which could increase the number of identifiedproteins. As a result, it was determined that appropriate sample sizesof human and mouse brains were respectively N=5 and N=3.

Note that whether or not all the samples formed normal distribution wasnot confirmed. Nevertheless, in the process of calculating the P-valuewith a computer, low-quality measurement results producing abnormalvalues were excluded in the present analysis using thecommercially-available program (ProteinPilot).

Additionally, the results obtained by the animal behavioral tests andtwo-photon microscope observation were basically analyzed by Student'sindependent t-test (two-sided test) in the sample sizes shown in figuresand descriptions thereof.

Moreover, brain tissue sampling, data collection in the massspectrometry, and the systems biology analysis were performed byindependent researchers without assigning the tasks to groups who knewthe circumstances.

Example 1

<Phosphoproteome Analysis on Alzheimer's Disease>

It has been suggested that various phosphorylation signal transductionsincluding tau phosphorylation are involved in a pathology of Alzheimer'sdisease. Accordingly, identifying phosphorylation signal transductionsin Alzheimer's disease, particularly, a phosphorylation signaltransduction which played a central role in a pre-onset stage ofAlzheimer's disease, makes it possible to provide very effective targetmolecules in early-stage diagnosis and treatment of this disease. Hence,the present inventor made efforts to comprehensively analyze(phosphoproteome analysis) phosphorylation signal transductions inAlzheimer's disease to identify a phosphorylation signal transductionwhich played a central roles in the pathology.

However, a postmortem change in protein phosphorylations basically quitehinders a phosphoproteome analysis targeting human postmortem brainsamples. In fact, the present inventor and other researchers haveheretofore performed proteome-wide analyses, in the postmortem humanbrain analysis, on the change in phosphoproteins of mouse brains storedat room temperature or 4° C. for different durations to determine aperiod during which the brain would reflect the living state beforedeath. As a result, the present inventor and other researchers haverevealed that various phosphoproteins had already changed at a timepoint 12 hours after the preservation was started (Oka, T., Tagawa, K.,Ito, H. & Okazawa, H. “Dynamic changes of the phosphoproteome inpostmortem mouse brains,” PLoS One, 2011, 6, e21405).

Hence, in view of this result, it was considered risky to conduct aphosphoproteome analysis based solely on human samples. Thus, effortswere made to identify phosphoproteins whose expression amounts changedin Alzheimer's disease, by the following stepwise approach: first,analyzing Alzheimer's disease model mice; and then analyzing brainsamples of Alzheimer's disease patients.

To be more specific, first, the following five types of transgenic mice(four types of AD model mice and one type of Tau model mice) weredissected at the ages of 1, 3, and 6 months (4, 12, and 24 weeks old).Then, the cerebral cortex, hippocampus, and striatum were quicklyseparated under a microscope and frozen immediately. Note that all ofthese processes were completed within 5 minutes, as assessed bymeasuring the time with a stopwatch.

(1) PS1 transgenic mice(2) PS2 transgenic mice(3) Human double-mutant APP695 transgenic mice(4) 5×FAD mice(5) Tau transgenic mice.

Additionally, the background of the APP-Tg2576 mice and the 5×FAD micewas B6/SJL, the background of the PS1 transgenic mice and the PS2transgenic mice was C57BL6, and the background of the Tau transgenicmice was C57BL6/C3H. Hence, these background mice were utilized ascontrol mice in the following experiment.

Note that, in the phosphoproteome analysis, preliminary tests by ABSCIEX Triple TOF 5600 mass spectrometry were repeated to determine theoptimal conditions allowing the detection of the largest number ofphosphoproteins. Moreover, samples were fractionated in multiple stagesusing cation exchange columns and reverse-phase columns. Data obtainedby combined analyses on the same samples were merged. As a result,conditions (appropriate amount, run time) for mass spectrometry allowingsuch quite a high detection score of confidence 95% were obtained.

Next, using these conditions, a phosphoproteome analysis was performedon the Alzheimer's disease model mice. To be more specific,phosphoproteins were purified from the five types of transgenic mice andthe three types of background mice corresponding thereto usingTITANSPHERE(registered trademark) Phos-Tio Kit. Then, after labelingwith eight different probes using the iTRAQ reagent, the analysis wasperformed by a single run of the mass spectrometry. Subsequently, thesystems biology analysis was performed based on the experimental resultsof three mass spectrometry analyses. As a result, with 95% confidence,744 to 1128 phosphoproteins were identified, and 13017 to 29995phosphopeptides were identified.

Thereafter, the proteins identified by nine mass spectrometry analyseswere mapped on the integrated protein-protein interaction (PPI) databaseusing a super computer. The utilized integrated database was the genomenetwork platform (http://genomenetwork.nig.ac.jp/index_e.html) providedby National Institute of Genetics. The integrated database includes theexperimentally-supported PPI database of the Human Genome Project (GNP),BIND (www.bind.ca/), BioGrid (www.thebiogrid.org/), HPRD(www.hprd.org/), IntAct (www.ebi.ac.uk/intact/site/index.jsf), and MINT(http://mint.bio.uniroma2.it/mint/Welcome.do).

After that, the mapped phosphoproteins were designated as nodes.Further, proteins linked to significantly changed phosphoproteins wereattached as accessory nodes. Moreover, links between the proteins wereconnected by lines (edges). Thus, a mouse default network was prepared.Note that, in this network, proteins indirectly linked to the identifiedproteins via two or more edges were excluded from the network.

Next, although one protein had multiple P-values of multiple peptidesderived therefrom, these P-values were integrated by the inverse normalmethod, and the integrated P-value was compared between the model micegroup and the control mouse group (n=3). Then, as a result of thecomparison between the AD model mice or Tau model mice at the ages of 1to 6 months and their control mice, changed phosphoproteins wereselected as nodes (p<0.05). Thus, although unillustrated, networks ofthe phosphoproteins changed at each time point of each model mouse wereconstructed.

Next, based on the constructed network of each model mouse thusobtained, phosphorylation signal transductions commonly changed in thesemodel mice were identified using two different approaches describedbelow.

(1) The First Approach

This is a selection approach based on a result of a simple comparison ofphosphoproteome data from multiple model mice at the same time point(hypothesis free approach)

(2) The Second Approach

This is a selection approach based on an assumption that an abnormalphosphorylation signal is generated in some process of amyloidaggregation or before the aggregation (Aβ aggregation-linked approach).

Note that, in the first approach, the number of common nodes among thedifferent models decreased with an increase in the number of AD modelscompared. To be more specific, the number of common nodes among the fourtypes of AD models was only one (only MAP1B at the age of 1 month),while no node was included at the ages of 3 and 6 months. Thus, forfurther analyses, although unillustrated, 65 nodes were selected whichwere common between two types of the AD model mice one or more times.Among the 65 proteins, there were 51 proteins whose phosphorylationschanged commonly in one combination of two types of the AD model mice ata certain single time point, while 14 proteins were phosphoproteinscommonly changed at multiple time points and phosphoproteins commonlychanged in multiple combinations of two types of the AD model mice(regarding the 14 proteins, see FIG. 1 ).

Meanwhile, in the second approach, an immunohistological analysis wasperformed on the four types of the AD model mice. Then, pathologicaldifferences were confirmed among these model mice. To be more specific,although unillustrated, in each of the 5×FAD mice and the APP mice,amyloid deposition started at the ages of 3 and 6 months. On the otherhand, in the PS1 mice and the PS2 mice, no amyloid deposition wasconfirmed even at the age of 6 months.

Thus, based on such a result, it was presumed that the 5×ADD mice andthe APP mice should share the pathological signal transduction at thetime when the Aβ deposition started. Hence, significantly changedphosphoproteins were compared between the 1-month-old 5×FAD mice and the3-month-old APP mice, or between the 3-month-old 5×FAD mice and the6-month-old APP mice.

Then, from this comparison result, 11 nodes common in the 5×FAD mice andthe APP mice were selected as phosphoproteins deduced to be linked to Aβaggregation in the brain (regarding the 11 nodes (proteins), see FIG. 1).

Surprisingly, as apparent from the result shown in FIG. 1 , collatingthe results obtained by the different approaches showed that eight ofthe 11 proteins selected by the Aβ aggregation-linked approach wereproteins also selected by the hypothesis free approach. On the otherhand, more than a half of the 14 proteins whose phosphorylation stateswere observed to be commonly changed at multiple time points between twotypes of the AD model mice were also selected by the Aβaggregation-linked approach.

Moreover, all of these phosphoproteins changed before the onset. To bemore specific, although unillustrated, the four types of the AD modelmice (PS1, PS2, APP, and 5×FAD) were subjected to the behavioral tests(Morris water maze test, rotarod test, open-field test, elevated plusmaze test, light-dark box test, and fear-conditioning test), but anyabnormal behavior was not detected at the age of 6 months.

As described above, in this phosphoproteomics analysis, the twoindependent approaches arrived at the similar conclusion, and 17phosphoproteins were identified as factors involved in a pre-onset stageof Alzheimer's disease and composing a network (AD core network) whichplayed a central role in the pathology.

Example 2

<AD Core Network Analysis in Tau Model Animals>

Although no conclusion has been drawn yet regarding the Alzheimercausative factor and onset mechanism, the most likely mechanism is suchthat when amyloid β molecules aggregate (amyloid pathology), theaggregation promotes tau phosphorylation and polymerization (taupathology), consequently leading to nerve cell death and so forth(amyloid cascade hypothesis). Hence, regarding the transition fromamyloid pathology to tau pathology presumed in this hypothesis, the 17proteins selected from the amyloid-pathology-induced AD model mice werere-analyzed targeting tau-pathology-induced AD model mice.

To be more specific, the 14 proteins also selected from the AD modelmice by the hypothesis free approach were compared with phosphoproteinswhich changed in the Tau model mice. As a result, it was revealed asshown in FIG. 34 that 10 phosphoproteins (ADDB, NFH, NFL, SPTA2, BASP1,CLH, MARCS, NEUM, SRRM2, and Marcksl1) were commonly changed between theAD model mice and the Tau model mice.

Moreover, although unillustrated, tau was not included in the 17proteins but included in the 65 proteins detected by the hypothesis freeapproach. To be more specific, the amount of the phosphorylated tauprotein was enhanced commonly in severe AD model mice (5×FAD and APP)and tau model mice at the age of 1 month, supporting that thephosphorylation of the tau protein linked amyloid pathology to taupathology.

Example 3

<AD Core Network Analysis in Human AD Patients>

Next, the 17 proteins selected from the result of the AD model micedescribed above were evaluated based on phosphoproteome data on thebrains of human AD patients.

To be more specific, first, in order to obtain phosphoproteome data onthe brains of human AD patients, mass spectrometry was performed usingfive brains of human AD patients as in the case of the mice. Note thatthe brain samples used in this analysis were isolated, frozen, andstored within 1 hour after death. The temporal lobe (temporal pole) andoccipital lobe (occipital pole) samples of the brains were subjected tothe analysis. Normally, these brain regions are remarkably affectedregions in AD patients. In addition, these brain samples were analyzedafter confirmed by a pathological examination that the samples were notcontaminated with tissues exhibiting no AD pathology, such as lewybodies and argyrophilic grains. Further, five brains derived fromhealthy subjects matching with the AD patients in age and five brainsderived from patients having dementia with lewy bodies were also used ascontrols in the analysis.

Although unillustrated, based on phosphoproteins detected in all thehuman brains as a result of the analysis, a human default network wasprepared. Note that, in this case, edges and accessory nodes were addedto nodes based on not only the human PPI database but also mouse and ratPPI databases so as not to miss important molecules such as kinases andphosphatases in comparing the human and mouse networks in the subsequentanalysis stage.

Then, each of the temporal lobe and occipital lobe was compared betweenthe AD patient brains and the normal brains or DLB patient brains, andchanged phosphoproteins were selected as “human (AD)-(normal) nodes” or“human (AD)-(DLB) nodes”. It was noteworthy that all thedisease-specific nodes in the “human (AD)-(DLB) nodes” were alsodetected in the “human (AD)-(normal) node” network.

Subsequently, networks were prepared based on the “human (AD)-(normal)nodes” in the temporal lobe and occipital lobe, and compared with theabove-described 17 proteins based on the mouse phosphoproteome. Theresult revealed as shown in FIG. 34 that ADDB, NFH, NFL, SPTA2, BASP1,G3P, MARCKS, and NEUM among the 17 proteins composing the AD corenetwork were changed commonly in the human AD patients.

Moreover, these nine phosphoproteins commonly changed also in the humanAD patients were identified as the phosphoproteins having been changedin the Tau model mice, except for G3P.

Thus, the above analysis results of the Tau model animals and the humanAD patients verified that the 17 proteins or at least most of them wereinvolved in a pre-onset stage of Alzheimer's disease and were componentsof a network which played a central role in the pathology.

Example 4

<Functional Analysis on AD Core Network>

Based on the integrated human-mouse PPI database described above, the ADcore network composed of the 17 proteins was prepared. FIG. 2 shows theobtained result.

As apparent from the result shown in FIG. 2 , surprisingly, 12 proteinsin the 17 proteins were directly linked, and three proteins (SRRM2,BASP1, and ADDB) were linked via one independent protein. Note that, inthe PPI database, Marcksl1 was a protein not directly linked to theother 16 proteins but exhibiting a high homology with MARCKS. Thisrevealed that at least 15 proteins formed a single functional network.

Further surprisingly, their functions were mainly directly related tosynapse functions such as spine formation, vesicle recycling, and energyproduction.

SPTA2 (brain α spectrin) is a protein cross-liked to actin and expressedat a high level in the brain (see Leto, T. L. et al., Mol. Cell. Biol.,1988, Vol. 8, pp. 1 to 9). It is known that SPTA2 interacts with SHANKat the postsynaptic density (see Bockers, T. M. et al., J. Biol. Chem.,2001, Vol. 276, pp. 40104 to 40112), and interacts with ADDB/adducin-b,one of the 17 proteins identified this time, formingspectrin/adducin/actin complexes (see Li, X. et al., J. Biol. Chem.,1996, Vol. 271, pp. 15695 to 15702). Moreover, it has also been revealedthat these proteins are substrates of protein kinase C (PKC), and afterthe phosphorylation of these proteins, the complex becomes unstable,decreasing the membrane stability, too.

It is known that ADDB contains a MARCKS-related domain, and that thephosphorylation by PKC controls the postsynaptic localization andinhibits actin/spectrin complex formation as described above (seeMatsuoka, Y. et al., J. Cell Biol., 1998, Vol. 142, pp. 485 to 497).Additionally, this system has been shown to control synapse productionand removal, although the result was at the neuromuscular junction (NMJ)in Drosophila (see Pielage, J. et al., Neuron, 2011, Vol. 69, pp. 1114to 1131).

MARCKS, BASP1, and NEUM are known to be greatly involved in signalsoriginating from lipid rafts. Additionally, MARCKS is a PKC-specificsubstrate and normally localized at the cell membrane. However, it isknown that after phosphorylated or bound to calmodulin, MARCKS isreleased from the cell membrane and transferred to the cytoplasm,inhibiting F-actin cross-linking (see Hartwig, J. H. et al., Nature,1992, Vol. 356, pp. 618 to 622). Further, various morphological andfunctional abnormalities have been observed in mouse brains havingmutant MARCKS (see Stumpo, D. J. et al., Proc. Natl. Acad. Sci. U.S.A.,1995, Vol. 92, pp. 944 to 948).

MRP/Marcksl1 is a member of the MARCKS family, and involved in PKCsignal transduction. Additionally, it is known that Marcksl1 isphosphorylated by JNK and controls the actin stability and thefilopodium formation of neurons (see Bjorkblom, B. et al., Mol. Cell.Biol., 2012, Vol. 32, pp. 3513 to 3526).

NEUM/neuromodulin/GAP43 is an important component of growth cone/axonpresynaptic terminals and is known as a main substrate of PKC (seeBenowitz, L. I. et al., Trends Neurosci., 1997, Vol. 20, pp. 84 to 91).Additionally, it is known that NEUM interacts with various molecules.For example, there is a report that NEUM interacts with PIP2 andpalmitate, or cytoskeletal proteins such as actin, spectrin,synaptophysin, and tau. As in the case of MARCKS and SPTA2, it has beenshown that NEUM is also controlled by calmodulin and moves between themembrane and cytoplasm (see Gamby, C. et al., J. Biol. Chem., 1996, Vol.271, pp. 26698 to 26705). As described above, NEUM is an adaptor proteinwhich controls presynaptic terminal functions via cytoskeletalregulation, and is suggested to be involved in memory and LTP formation(see Routtenberg, A. et al., Proc. Natl. Acad. Sci. U.S.A., 2000, Vol.97, pp. 7657 to 7662).

BASP1/NAP-22/CAP23 is myristoylated protein having a PEST motif, and isabundant in axonal terminals (see Mosevitsky, M. I. et al., Biochimie,1997, Vol. 79, pp. 373 to 384). Although the function has not beensufficiently elucidated, BASP1/NAP-22/CAP23 exists at the inner surfaceof a lipid raft in the cell membrane. In addition, BASP, MARCKS, andNEUM seem to regulate PI(4,5)P2 by a common mechanism. Further, it issuggested that the phosphorylation-dependent interaction betweencalmodulin and BASP, MARCKS, or NEUM promotes actin network formation(see Laux, T. et al., J. Cell Biol., 2000, Vol. 149, pp. 1455 to 1472).

As described above, it is suggested that the proteins composing the ADcore network form a network which controls presynaptic and postsynaptocmorphologies.

Meanwhile, phosphoproteomic changes of SYT1/synaptotagmin 1 and GPRIN1/Gprotein regulated inducer of neurite outgrowth 1 were not detected inthe brains of both the Tau model mice and the human AD patients.Nevertheless, these proteins might also be involved in a pre-onset stageof AD. Particularly, SYT1/synaptotagmin 1 is important because itcontrols vesicle recycling at synaptic terminals. Note that SYT1 isknown to form a complex at synaptic terminals with a vesicle cargomolecule CLH/clathrin heavy chain selected by both of the hypothesisfree approach and the Aβ aggregation-linked approach (see Schwarz, T.L., Proc. Natl. Acad. Sci. U.S.A., 2004, Vol. 101, pp. 16401 to 16402).Additionally, GPRIN1 is a Gao effector enriched in the growth conemembrane fraction which induces neurite outgrowth (see Chen, L. T., J.Biol. Chem., 1999, Vol. 274, pp. 26931 to 26938). However, the detailedfunctions have not been elucidated yet.

Further, interestingly, molecules involved in energy production werealso selected. To be more specific, ATPA and ATPB are mitochondrial ATPsynthase subunits A and B, respectively. ACON is mitochondrial aconitase2 which catalyzes citrate isomerization in TCA cycle. Moreover,G3P/GAPDH/glyceraldehyde-3-phosphate dehydrogenase is an importantenzyme in glycolysis, also plays a nuclear function through thenitrosylase activity, and affects microtubule assembly by the samemechanism. Thus, the enzyme is also related to cytoskeleton.

SRRM2 is involved in splicing together with SRm160 (see Blencowe, B. J.,Genes Dev., 1998, Vol. 12, pp. 996 to 1009), and is functionallydifferent from the other core network proteins.

HS90A/HSP90 is a chaperon molecule involved in quality control andfolding of various proteins. Because of the general roles, HS90A/HSP90is linked to various proteins in the core network.

As described above, it was revealed that the phosphoproteomic changes inthe pre-onset stage of Alzheimer's disease were selectively focused ontwo or three networks which controlled synapse function and energymetabolism.

<Changes in Phosphoproteins Due to Aging in AD Model Animals>

Further analyzed was how chronological changes in phosphoproteins(changes in phosphoproteins by pathological aging) in AD model mice wererelated to those (chronological changes in phosphoproteins in thebackground mice) in normal aging (physiological aging). Note thatproteins from which data were not obtained with a high confidence at anytime point of the 5×FAD mice were excluded from this analysis.

In this analysis, three sets of new samples including the 5×FAD mice andthe background mice at the ages of various time points from 1 to 12months were analyzed by the mass spectrometry. Moreover, based on valuesof the 1-month-old background mice obtained regarding the 17phosphoproteins, values of the 5×FAD mice and the background mice at theages of each month were corrected and plotted on a graph to analyze thechronological changes in these phosphoproteins.

As a result, although unillustrated, the patterns of changes in most ofthe phosphoproteins composing the AD core network were qualitativelysimilar between the physiological aging and the ageing due to thepathological aging. However, the patterns were quantitatively different.It was revealed that each phosphoprotein had a time point when adifference in the expression amount thereof was remarkably large betweenthe 5×FAD mice and the background mice.

For example, regarding MARCKS, Marcksl1, and SRRM2, amounts of thesephosphoproteins in the 1-month-old 5×FAD mice were remarkably large incomparison with those of the background mice. Note that the differencewas diminished over time. Moreover, regarding SPTA2, G3P, ADDB, SYT1,BASP1, HSP90A, and NEUM, amounts of these phosphoproteins in the3-month-old 5×FAD mice were remarkably large in comparison with those ofthe background mice. Further, regarding NFH, NFL, CLH, and GPRIN1,amounts of these phosphoproteins in the 12-month-old 5×FAD mice wereremarkably different from those of the background mice.

Furthermore, based on the result of analyzing the above chronologicalchanges in the phosphoproteins, the core protein network wasreconstructed. FIG. 3 shows the obtained result.

As described above, the phosphorylations of MARCKS and its homologMarcksl1 changed at the initial phase. Moreover, it was revealed asshown in FIG. 3 that the changes of these were followed by those of theother core phosphoproteins belonging to the same functional domain orrelated functional domains.

Example 5

<Search for Kinases/Phosphatases Involved in Change in AD Core Network>

As shown in FIG. 3 , the chronological changes in the phosphorylationsof the proteins composing the AD core network in the pre-onset stage ofAlzheimer's disease can be categorized into three patterns: one having apeak at the initial phase, one having a peak at the mid phase, and onehaving a peak at the late phase. Moreover, in view of these categories,it is presumed that the phosphorylations of the proteins composing theAD core network are controlled by particular kinases and the like in atime-specific manner.

Hence, in order to search for kinases and the like involved in this ADcore network control, first, kinases and phosphatases were selectedamong the proteins directly linked to the proteins composing the AD corenetwork. Then, among these kinases and phosphatases, PKC was consideredas the most important enzyme involved in the change in the AD corenetwork from the viewpoint that it could phosphorylate the largestnumber of the core protein. Note that, as having been already revealedfrom various reports in the past regarding AD pathology, MAPK and MAPKKKwere identified after PKC.

Moreover, as the second group following these three kinases, identifiedwere casein kinase (CSKII), receptor-interacting proteinserine/threonine kinase 1/3 (RIP1/3), cyclin-dependent kinase 5/6(CDK5/6), and protein kinase C-like protein 1 (PKN1). Further, as thethird group, identified were Ca2+/calmodulin-dependent kinase(CaMKI/II), protein kinase D (PKD), and the like. Moreover,Lck/Yes-related novel protein tyrosine kinase (Lyn) and other 20 kinaseswere identified as candidates to control the core network, althoughthese were linked to just one core protein.

FIG. 3 shows the enzyme-substrate relation obtained by comparing theresult of analyzing the above kinases linked to the core phosphoproteinswith the result of the chronological changes in the core phosphoproteinsin the 5×FAD mice described above.

As apparent from the result shown in FIG. 3 , the initial phase (at theage of 1 month), the mid phase (at the age of 3 months), and the latephase (at the age of 6 months) patterns of the core phosphoproteins wereobserved to have correlations with PKC, Lyn, CamK, and CASK.

It seemed that the PKC family among these activated various kinasesearliest, and that the activation continued until the late phase. To bemore specific, it was revealed that first the PKC family phosphorylatedMARCKS and Marcksl1, and next the kinase family phosphorylated G3P,NEUM, BASP1, and SPTA2.

Further, in order to identify target sequences of the kinases whichcontrolled 18 proteins composing the AD core network, the data on thepeptide phosphorylations obtained from the mass spectrometry wereexamined again, and a phosphorylation level at one site of each proteinwas individually analyzed. To be more specific, identified werepolypeptides containing one phosphorylation site whose amount changed incomparison with the wild type at P<0.05 at one or more time points in atleast one model among the five types of transgenic mice (the four typesof AD model mice and one type of Tau model mice).

As a result, although unillustrated, significant changes in thephosphorylation levels were observed in ADDB at serine at position 60,serine at position 62, serine at position 532, serine at position 594,serine at position 602, serine at position 618, serine at position 692,and serine at position 700. Significant changes in the phosphorylationlevels were observed in NFH at serine at position 500, serine atposition 535, serine at position 583, serine at position 673, serine atposition 721, serine at position 763, serine at position 795, serine atposition 834, threonine at position 839, serine at position 867, andserine at position 888. Significant changes in the phosphorylationlevels were observed in NFL at serine at position 473, serine atposition 523, and serine at position 532. Significant changes in thephosphorylation levels were observed in SPTA2 at serine at position 1031and serine at position 1217. Significant changes in the phosphorylationlevels were observed in ATPB at threonine at position 262 and threonineat position 453. Significant changes in the phosphorylation levels wereobserved in BASP1 at threonine at position 31, threonine at position 36,serine at position 92, serine at position 131, serine at position 192,and serine at position 218. Significant changes in the phosphorylationlevels were observed in G3P at threonine at position 182 and threonineat position 209. Significant changes in the phosphorylation levels wereobserved in GPRIN1 at serine at position 182, serine at position 219,serine at position 495, serine at position 576, serine at position 691,serine at position 693, serine at position 714, serine at position 764,serine at position 771, serine at position 816, and threonine atposition 795. Significant changes in the phosphorylation levels wereobserved in MARCKS at serine at position 26, serine at position 27,serine at position 29, serine at position 113, serine at position 122,serine at position 124, serine at position 125, serine at position 127,serine at position 128, serine at position 138, serine at position 140,serine at position 141, threonine at position 143, serine at position163, serine at position 171, and serine at position 299. Significantchanges in the phosphorylation levels were observed in NEUM at serine atposition 86, threonine at position 89, serine at position 96, serine atposition 142, threonine at position 172, and serine at position 193.Significant changes in the phosphorylation levels were observed in SRRM2at serine at position 1067, serine at position 1278, serine at position1305, serine at position 1339, serine at position 1359, serine atposition 1360, serine at position 2351, serine at position 2084, serineat position 2404, serine at position 2535, threonine at position 1448,and threonine at position 2350. Significant changes in thephosphorylation levels were observed in Marcksl1 at serine at position22, threonine at position 85, serine at position 104, threonine atposition 148, serine at position 189, serine at position 151, and serineat position 185. Significant changes in the phosphorylation levels wereobserved in HS90A at serine at position 231 and serine at position 263.Significant changes in the phosphorylation levels were observed in SYT1at threonine at position 125 and threonine at position 128.

<Involvement of Kinases in Alzheimer's Disease Pathology>

The significance of the AD core network, which was constructed based onthe above-described analysis result, in the pathology of Alzheimer'sdisease was verified using in vivo and in vitro experimental systems.

The functions of the core factors revealed by the phosphoproteomeanalysis suggested that specific phosphorylation signals linkingpresynaptic cytoskeleton to postsynaptoc cytoskeleton were perturbed atthe earliest stage before the onset of Alzheimer's disease.Particularly, it has been suggested that the cytoskeleton networkcomposed of actin binding proteins such as actin and spectrin mainlycontrols dendritic spine formation (see Matus, A., Science, 2000, Vol.290, pp. 754 to 758, Tada, T. et al., Curr. Opin. Neurobiol., 2006, Vol.16, pp. 95 to 101). Hence, it was presumed that the link from MARCKS toactin polymerization and the link from SPTA2 to actin-spectrincross-linking became abnormal at the initial phase of Alzheimer'sdisease, and that these cytoskeleton network activations affected thedendritic spine dynamics, and were consequently involved in thepathology of Alzheimer's disease.

Thus, the dendritic spine dynamics were analyzed with a two-photonmicroscope targeting the 5×FAD mice (12 weeks old) before the onset ofAlzheimer's disease. FIGS. 4 to 9 show the obtained result.

Living cortical neurons of layer 1 of the 12-week-old 5×FAD mice wereobserved with a two-photon microscope. The result revealed as shown inFIGS. 4 and 5 that the dendritic spine densities of the cortical neuronswere remarkably decreased. Moreover, decreases in the number of spinesper dendritic shaft length were observed in all the spine types (thin,mushroom, and stubby) (see FIG. 6 ). Further, regarding absolute numbersof dendritic spines per 100 μm, all of the formed spines, eliminatedspines, and stably remaining spines were decreased (see FIGS. 7 to 9 ).Moreover, regarding the percentages of three types of spine dynamics inthe 5×FAD mice, the formed spines were decreased. On the other hand, noremarkable change was observed in the eliminated spines and the stablyremaining spines (see FIG. 9 ). In sum, it was revealed that the spineformation in process was affected in the pathology of the 5×FAD mice.Note that these results basically agree with the findings in the pastregarding AD model mice (see Palop, J. J. et al., Neurosci., 2010, Vol.13, pp. 812 to 818, Wei, W. et al., Nat. Neurosci., 2010, Vol. 13, pp.190 to 196, Wu, H. Y. et al., J. Neurosci., 2010, Vol. 30, pp. 2636 to2649).

Hence, next, kinases were analyzed which were suggested to affect the ADcore network from the initial phase to the mid phase of the pre-onsetstage of Alzheimer's disease. More concretely, the effect of the kinaseson the synaptic pathology in the 5×FAD mice was analyzed byadministering inhibitors and so forth against these kinases into thesubarachnoid spaces of the mice. FIGS. 4 to 9 show the obtained result.

Administering a PKC inhibitor (Go6976) ameliorated spine abnormality inthe 5×FAD mice and increased the total spine density (see FIGS. 4 and 5). Moreover, the numbers of spines of thin, mushroom, and stubby typeswere also increased (see FIG. 6 ). Further, regarding the spinedynamics, the Go6976 treatment enhanced the spine formation andstability (see FIGS. 7 to 9 ).

Moreover, it was revealed that the treatment with a CamKII inhibitor(KN-93) also had a therapeutic effect on the spine formation andstability. Further, it was also revealed that the numbers of all thetypes of dendritic spines that had been decreased in the 5×FAD mice wererecovered by the treatment (see FIGS. 7 to 9 ).

Further, it was revealed that the treatment with a Lyn kinase activator(MLR1023) also had a therapeutic effect on the spine formation andstability as in the case of the treatment with the PKC inhibitor orCamKII inhibitor (see FIGS. 7 to 9 ). Note that MLR1023 found to beeffective this time is also known as an insulin receptor potentiatingagent. Hence, the therapeutic effect this time is presumably also aneffect of alleviating the insulin resistance, which is observed in thebrains of Alzheimer's disease patient (see Craft, S., Nat. Rev. Neurol.,2012, Vol. 8, pp. 360 to 362).

Furthermore, to confirm the effects of the kinase inhibitors in an invivo system, an immunohistological analysis was performed targetingbrain samples after two-photon microscope observation, and usingantibodies against kinases in activated forms. FIGS. 10 and 11 show theobtained result.

As a result, it was verified as shown in FIGS. 10 and 11 that PKCβ,PKCδ, and CamKII (CamKIIα) were activated in the cortical areas(retrosplenial cortexes) of the 5×FAD mice. Moreover, the result ofquantifying signals generated from immunostained sites confirmed theabove-described effects of the kinase inhibitors.

<Involvement of MARCKS in Spine Pathology of Alzheimer's Disease>

As described above, MARCKS was detected as a protein whosephosphorylation changed at the initial phase of Alzheimer's disease bythe aging pattern analysis and also by the aggregation-linked approachdescribed above (see FIG. 34 and FIG. 3 ). This suggests that MARCKS isthe most reliable phosphorylation signal transduction molecule in thepre-onset stage of the pathology of Alzheimer's disease.

To confirm this, MARCKS, which is a substrate of PKC and CamKII, wasknocked down in AD model mice. To be more specific, a lentiviral vectorexpressing a shRNA against MARCK was injected into the cortexes of the5×FAD mice (12 weeks old) before the onset. FIGS. 12 to 18 show theobtained result.

As apparent from the result shown in FIGS. 12 to 18 , the spinepathology in the 5×FAD mice was ameliorated by suppressing theexpression of MARCKS with the shRNA. Moreover, the shRNA against MARCKSrecovered the decrease in the number of spines, increased the numbers ofimmature and matured spines, and further improved the spine formation inthe 5×FAD mice.

Example 6

<Search for Kinase Involved in Transition from Amyloid Pathology to TauPathology>

In addition to the identification of the specific network in thepre-onset stage of Alzheimer's disease described above, efforts weremade to identify a kinase capable of promoting tau phosphorylation.

The proteins selected based on the hypothesis free approach describedabove did not directly include kinases/phosphatases, but the enhancementof a b-raf protein amount was observed in two 3-month-old AD model mice(p<0.05). Morover, the enhancement was observed in three 1-month-old ADmodel mice and two 6-month-old AD model mice, too.

Hence, whether or not inhibiting b-raf actually enabled regulation oftau pathology was tested. To be more specific, an amyloid β protein(Aβ1-42) and three types of b-raf inhibitors were added to primarycultures of cortical nerve cells, and tau phosphorylation in thecortical nerve cells was analyzed. Note that it has been revealed thattreating cortical nerve cells with Aβ enhances tau phosphorylation.FIGS. 19 and 20 show the obtained result.

As apparent from the result shown in FIGS. 19 and 20 , the tauphosphorylation was suppressed in the Aβ-treated cortical nerve cells.Moreover, although unillustrated, the enhancement of tau phosphorylationwas commonly observed in severe AD model mice (5×FAD, APP) and tau modelmice at the age of 1 month. To be more specific, this means the tauphosphorylation surprisingly starts in the brains of young AD model mice(1 month old) before immunohistological and symptomatic changes.

Thus, the above result suggested that b-raf was involved in thepromotion of the transition from amyloid pathology to tau pathology.

—Frontotemporal Lobar Degeneration—

In the present Examples, next, analyses were performed by employingexperimental methods and so forth described below to identify a signaltransduction pathway which played a central role in a pre-onset stage offrontotemporal lobar degeneration, and consequently to provide targetmolecules useful in the diagnosis and treatment of frontotemporal lobardegeneration. Additionally, experiments other than those specificallydescribed below were conducted as in the case of the above Alzheimer'sdisease analyses, unless otherwise specifically stated.

<Frontotemporal Lobar Degeneration Model Mice>

In the present Examples, frontotemporal lobar degeneration model micewere prepared to search for the target molecules.

It has been known that arginine at position 504 of the PGRN(progranulin) protein is conserved across species, and that a pointmutation at this site mainly causes dementia (see Nicholson, A. M. etal., Alzheimers Res. Ther. 4, 4 (2012), Le Ber, I. et al., Hum. Mutat.,2007, Vol. 28, pp. 846 to 855).

Hence, in order to introduce a stop mutation into this site,heterozygous PGRN-R504X mutation knockin mice were prepared. In thepreparation, a Neo cassette was inserted in C57BL/6J mice.

To be more specific, first, a targeting vector for the knockin micepreparation was constructed using the following two types of constructs.

(Construct 1)

A 6.5-kbp NotI-XhoI fragment with the R504X mutation was amplified byPCR from a Bac clone (ID: RP23-311P1 or RP23-137J17) and subcloned intoa pBS-DTA vector (manufactured by Unitech Co., Ltd.).

(Construct 2)

A 2999-bp ClaI-XhoI fragment was amplified by PCR and subcloned into apBS-LNL(−) vector (manufactured by Unitech Co., Ltd.) with the Neocassette.

Then, the BamHI (Blunt)-XhoI fragment derived from the construct 2 wasinserted between XhoI (Blunt)-SalI sites of the construct 1 andsubcloned. The vector thus obtained was used as a targeting vector.

Next, the targeting vector was linearized by SwaI treatment, and thenintroduced into ES clones of C57BL/6J mice by electropolation. Thegenotype analysis of the ES clones was performed by PCR. Positive clonesin this analysis were analyzed by Southern blotting using a probe forneomycin (Neo). Then, ES clones confirmed to have homologousrecombination occurred between the targeting vector and the genome ofthe C57BL/6J mice were injected into a mouse early embryo to preparechimeric mice. Subsequently, the chimeric mice were bred with CAC-Cremice to remove the Neo cassette in the F1 mouse genome. Further, usingthe genomic DNA prepared from the tail of the F1 mouse thus obtained,individuals in which the PGRN mutation was introduce by the knockin wereselected from these mice. Thus, PGRN-KI mice were established. Notethat, unless otherwise specifically stated, the PGRN-KI mice describedbelow refer to the heterozygotes.

Moreover, the PGRN-KI mice thus prepared were subjected to a westernblot analysis using an anti-PGRN antibody. It was confirmed that anamount of the full-length PGRN protein expressed was reduced in thecerebral cortexes of the mice. Further, as predicted from thenonsense-mediated RNA decay mechanism, the quantitative PCR confirmedthat an amount of the mutant mRNA expressed was also reduced in thePGRN-KI mice. Furthermore, PGRN of the PGRN-KI mice was immunostained,and the result was collated with that of a nerve-cell marker proteinNeuN. From this, it was suggested that the reductions of the PGRN inboth the cerebral cortex and the cerebellum were mainly attributable tothe reduction in nerve cells.

In addition, interestingly, the body weights of the PGRN-KI mice atbirth were lighter than those of mice having the same genetic backgroundused as a control (C57BL/6J, hereinafter also referred to as “backgroundmice”). Nonetheless, 20 weeks after birth, the body weights of most ofthe PGRN-KI mice were not much different from that of the wild type.Further, the weight of the brain was slightly light in comparison withthe control, but no structural abnormality was observed.

<Recovery Experiment with Vemurafenib and shRNA>

A pharmacological recovery experiment was conducted on the PGRN-KI miceas follows. To be more specific, an osmotic pump (1 μl/hour, 1003D,manufactured by Durect Corporation) was introduced into the subarachnoidcavity of a 12-week-old mouse, and 1.7 μM vemurafenib (S1267,manufactured by Selleckchem Chemicals) or PBS was supplied for 3 days.In addition, 0, 8, and 24 hours on Day 3 after the introduction, imagingwas performed.

Moreover, 3 μl of a shRNA-Tau lentiviral vector (sc-430402-V,manufactured by Santa Cruz Biotechnology Inc., 1×10⁶ TU) or scrambledshRNA (SC-108080, manufactured by Santa Cruz Biotechnology Inc., 1×10⁶TU) was injected into the same region as in the case of AAV1-EGFP(regarding AAV1-EGFP, see <In Vivo Imaging with Two-Photon Microscope>for Alzheimer's disease described above). In addition, 0, 8, and 24hours on Day 5 after the shRNA injection, imaging was performed.

<Analysis on Mislocalization of Tau Protein in Spine>

A coimmunostaining analysis was performed using an anti-PSD-95 antibodyand an anti-phosphorylated tau antibody (Ser203 or Thr220). To be morespecific, paraffin sections (5 μm) were prepared from retrosplenialcortex (RSD) tissues of the PGRN-KI mice and so on, co-stained with theantibodies, and observed by LSM510 confocal microscope (manufactured byZeiss, objective magnification: ×63, zoom 1, Z-stack images were set atintervals of 0.8 μm).

In the co-localization analysis, the number of sites wherephosphorylated tau and PSD-95 signals overlapped with each other wascounted in the obtained images (143 μm×143 μm).

Moreover, the fluorescent signal intensity derived from thephosphorylated tau or PSD-95 was quantified using ZEN lite 2012(manufactured by Zeiss). An average pixel intensity per ROI (20 μm×20μm) was calculated.

Then, data were obtained from randomly set 10 images, and used for thecomparison between the mouse groups by statistical analyses (one-wayanalysis of variance and Tukey's multiple comparison test).

Reference Example

<Phenotype Analysis on PGRN-KI Mice>

First, analyzed was whether or not the PGRN-KI mice having the stopmutation introduced in the PGRN (progranulin) gene as described abovewould exhibit the frontotemporal lobar degeneration (FTLD) phenotype.

PGRN-related FTLD is classified as FTLD-TDP characteristized by TDP43aggregates in the nucleus and cytoplasm. Note that TDP43 is a nuclearprotein involved in RNA processing, but the aggregate may be formed bythe cytoplasmic translocation.

Hence, brain tissues of the PGRN-KI mice were stained with an anti-TDP43antibody. As a result, although unillustrated, signal intensities of theTDP43 staining were not uniform in the frontal cortexes of the PGRN-KImice from the age of 1 month. Moreover, the number of nerve cells notstained or weakly stained with the anti-TDP43 antibody was apparentlyincreased in the PGRN-KI mice in comparison with that of the backgroundmice. Further, the difference became remarkable over time.

In addition, cytoplasmic inclusion, lentiform intranuclear inclusion,and cytoplasmic TDP43 staining were observed in the PGRN-KI mice.Further, in the mice, ubiquitin-positive aggregates were also observed.

These characteristics were pathological findings observed inPGRN-related FTLD of human. Thus, it was revealed that the PGRN-KI micewere pathologically similar to patients of this disease.

On the other hand, p62 and FUS inclusion, which are rarely observed inhuman pathology, were observed in the PGRN-KI mice as an atypicalfinding. These inclusions recognized by an anti-p62 antibody or ananti-FUS antibody were detected in the frontal cortexes (M2) at the ageof 4 months, and spread to the parietal cortexes at the age of 6 months.Meanwhile, in the PGRN-KI mice, an apparent increase in apoptosis, whichwould be detected by Tunel staining, was not observed until the age of12 months.

Further, in addition to the analysis on the protein aggregate, whetheror not an inflammation was activated in the PGRN-KI mice was analyzed.Note that, such inflammation activation is a characteristic commonlyobserved across many neurodegenerative diseases. An immunostaining wasperformed on the PGRN-KI mice using an anti-IBA1 antibody and ananti-GFAP antibody. The result revealed that inflammation was activatedin each of microglia and astrocytes. Moreover, such inflammationactivations were also confirmed by quantitative PCR targeting IL-1b andCox-2. However, the invasion of CD4- or CD8-positive cells was notobserved.

Further, another common characteristic of neurodegenerative diseasesincludes DNA damage. An apparent increase in γH2AX focus formation wasobserved in cortical nerve cells at the age of 6 months. This revealedthat DNA damage occurred in the PGRN-KI mice, too.

Furthermore, the PGRN-KI mice were also subjected to six behavioraltests as in the case of Alzheimer's disease model mice described above.As a result, abnormalities regarding anxiety memory and anxiety-relatedmemory were not observed in the open-field test, the light-dark boxtest, and the elevated plus maze test. Nevertheless, an apparentdecrease in memory formation was observed in the fear-conditioning test.Note that it can be said that this decrease in the memory formation wasnot due to a disorder in a sensory function or motor function because noabnormal score was observed in the rotarod test. Moreover, in the Morriswater maze test, a statistically significant decrease was observed fromthe age of 3 months in the time during which the mice stayed in thetarget region or the number of times the mice passed through the target.This result supported the loss of the memory formation in the PGRN-KImice. Note that, in the Morris water maze test using the PGRN-KI mice,the mice received the 60-second trial four times a day for 5 days tolearn the position of the platform (target region). Then, the test wasconducted under a condition where the platform was removed to measurethe time during which the mice stayed in the target region where theplatform was originally located and the number of times the mice passedthrough the target. Additionally, these characteristics observed in thebehavioral tests basically agree with clinical symptoms of FTLD patientshaving an R504 stop mutation which mainly develops dementia.

As described above, the PGRN-KI mice reflected both the pathologicalobservations and the clinical symptoms of FTLD patients, revealing thatthe mice were quite useful as FTLD model animals.

Example 7

<Phosphoproteome Analysis on FTLD>

Using the PGRN-KI mice whose usefulness as FTLD model animals wasverified, efforts were made, as in the case of the above Alzheimer'sdisease analyses, to comprehensively analyze (phosphoproteome analysis)phosphorylation signal transductions in FTLD also to identify aphosphorylation signal transduction which played a central role in apathology of the disease.

Particularly, PGRN has been reported to exhibit an antagonistic actionagainst TNF; on the other hand, contradictory results have also beenreported (see NPLs 17 to 21). To elucidate this contradiction, acomprehensive proteome analysis was performed on the cerebral cortextissues of the PGRN-KI mice to examine, in the brains of the PGRNmutation-related FTLD model mice, whether a TNF signal transductionpathway was activated or a different type of signal transduction pathwaywas activated.

To be more specific, the comprehensive proteome analysis was performedas in the case of Alzheimer's disease described above using ABSCIEX 5600and targeting the cerebral cortex tissues derived from three PGRN-KImice and those derived from three background mice (C57BL/6J).

Then, based on the comprehensive proteome data thus constructed, whetheror not the TNF signal transduction pathway was activated in the cerebralcortex tissues of the PGRN-KI mice was examined. Concretely, usingsignal transduction pathway-related database(http://www.genome.jp/kegg/pathway.html) of KEGG (Kyoto Encyclopedia ofGenes and Genomes), proteins belonging to the TNF signal transductionpathway were searched for proteins whose phosphorylation states changedin the PGRN-KI mice and C57BL/6J mice. As a result, surprisingly, noprotein whose phosphorylation changed was found in the TNF signaltransduction pathway per se for 1 to 6 months after birth and after theonset.

Hence, next, an analysis was performed targeting 16 TNF-related signaltransduction pathways including an adipocytokine signal transductionpathway, a NF-kB signal transduction pathway, and an apoptosis signaltransduction pathway. The result revealed that, in a MAPK signaltransduction pathway, an mTOR signal transduction pathway, and a signaltransduction pathway related to antigen processing and presentation,phosphorylations of proteins belonging to these signal transductionpathways remarkably changed in the PGRN-KI mice in comparison with theC57BL/6J mice.

Further, it was also revealed that, in these signal transductionpathways, the most remarkable change in the phosphorylation was focusedon the MAPK signal transduction pathway which would lead to tau proteinphosphorylation.

Note that the mTOR signal transduction pathway and the MAPK signaltransduction pathway were common in PKC activation. On the other hand,in the signal transduction pathway related to antigen processing andpresentation, the protein phosphorylation varied for 1 to 6 months afterbirth.

The MAPK signal transduction pathway was apparently activated in thePGRN-KI mice from the pre-onset stage. During the period of symptomprogression also, multiple proteins belonging to the signal transductionpathway were in high phosphorylation states all the time.

Particularly, in seven proteins, b-raf, PKCα, PKCβ, PKCγ, tau, MAP2K1(mitogen-activated protein kinase kinase 1, MAP kinase kinase 1, MEK-1),and stathmin belonging to the MAPK signal transduction pathway, thephosphorylations at one or two amino acid sites of each of theseproteins (phosphopeptide amounts detected by the mass spectrometry)remarkably changed in the PGRN-KI mice in comparison with the C57BL/6Jmice.

Importantly, the phosphorylation of the tau protein significantlychanged at multiple sites. Particularly, serine at position 203,threonine at position 220, and serine at position 393 of the tau protein(corresponding respectively to serine at position 214, threonine atposition 231, and serine at position 404 of human tau protein) were inhigh phosphorylation states all the time during the above-describedperiod, or the phosphorylations were enhanced over time.

Moreover, regarding b-raf and PKCγ also, one or multiple sites thereofwere in high phosphorylation states all the time, or thephosphorylations were enhanced over time.

More concretely, in the PGRN-KI mice, the phosphorylation of serine atposition 348 of b-raf was 1.1487 times, 1.1795 times, and 1.3664 times(shown are relative values at the ages of 1 month, 3 months, and 6months, respectively) as high as those of the C57BL/6J mice. Moreover,regarding serine at position 766 of b-raf, the phosphorylation was1.7508 times and 3.0476 times (shown are relative values at the ages of3 months and 6 months, respectively). Further, regarding serine atposition 769 of b-raf, the phosphorylation was 1.9752 times (shown is arelative value at the age of 6 months). Note that these serine atposition 348, serine at position 766, and serine at position 769 ofb-raf correspond respectively to serine at position 365, serine atposition 729, and serine at position 732 in human.

In addition, for PKCγ, in the PGRN-KI mice, the phosphorylation ofthreonine at position 655 was 1.2052 times, 1.1308 times, and 1.5702times (shown are relative values at the ages of 1 month, 3 months, and 6months, respectively) as high as those of the C57BL/6J mice. Moreover,regarding serine at position 690 of PKCγ, the phosphorylation was 2.5918times (shown is a relative value at the age of 1 month). Note that thesethreonine at position 655 and serine at position 690 of PKCγ correspondrespectively to threonine at position 655 and serine at position 690 inhuman.

Moreover, although unillustrated, regarding serine at position 766 ofb-raf and threonine at position 655 of PKCγ, it was confirmed by westernblotting that the phosphorylations at these sites in the cerebralcortexes of the 3-month-old PGRN-KI mice were enhanced in comparisonwith those in the 3-month-old C57BL/6J mice as in the above result ofmass spectrometry.

Further, the phosphorylation of MEK-1 (Map2k1) located downstream ofb-raf and PKCγ was also analyzed by western blotting. It was confirmedthat the phosphorylation in the cerebral cortexes of the PGRN-KI mice atthe age of 3 months was also enhanced in comparison with that of the3-month-old C57BL/6J mice.

Furthermore, an immunohistological analysis using an anti-phosphorylatedtau antibody AT8 was performed to detect phosphorylated tau protein inthe cytoplasms of the frontal lobe nerve cells of the PGRN-KI mice. As aresult, a signal of the phosphorylated tau protein was detected at theage of 12 months.

On the other hand, a signal transduction induced by TNF was analyzed bya co-immunoprecipitation method based on amounts of complexes formed inthe signal transduction (TNFR-TRADD complex, TNFR-RIP complex, andTNFR-TRAF2 complex). As a result, no significant difference was foundbetween the PGRN-KI mice and the C57BL/6J mice.

The above results revealed that, in the PGRN-KI mice, the MAPK signaltransduction pathway was activated, while the TNF signal transductionpathway was not activated.

Example 8

<Analysis on Therapeutic Effect of b-raf Inhibitor on BehavioralPhenotype of FTLD Model Mice>

Analyzed was whether or not suppressing an abnormal activation in theMAPK signal transduction pathway by using a b-raf specific inhibitorwould recover the behavioral phenotype of the PGRN-KI mice.

To be more specific, in accordance with the protocol shown in FIG. 21 ,a b-raf specific inhibitor vemurafenib or PBS was provided to the6-week-old PGRN-KI mice every day over 6 weeks. Then, the behaviors ofthese mice were evaluated in the Morris water maze test and thefear-conditioning test. FIGS. 22 and 23 show the obtained result.

As apparent from the result shown in FIGS. 22 and 23 , administeringvemurafenib to the PGRN-KI mice remarkably recovered the scores in thetwo tests.

Meanwhile, the therapeutic effect of thalidomide was also tested. Notethat thalidomide is known to suppress the TNF signal transductionpathway. Concretely, in accordance with the protocol shown in FIG. 24 ,thalidomide was administered to the PGRN-KI mice by peritoneal cavityinjection every day for the same period as that in the protocol usingthe b-raf inhibitor. The behaviors of these mice were evaluated by theabove two tests. FIGS. 25 and 26 show the obtained result.

As apparent from the result shown in FIGS. 25 and 26 , in thethalidomide administration example also, beneficial effects were finallyverified regarding the scores in the Morris water maze test and thefear-conditioning test.

Note that such symptom recoveries were not observed in thePBS-administered PGRN-KI mice.

Moreover, the cerebral cortexes of the PGRN-KI mice treated with theagent were analyzed by western blot. The result confirmed as shown inFIG. 27 that vemurafenib suppressed the b-raf phosphorylation. Further,it was revealed that vemurafenib also suppressed the PKCphosphorylation.

On the other hand, in the thalidomide administration example, the b-rafphosphorylation was suppressing, but the PKC phosphorylation was notsuppressed, as apparent from the result shown in FIG. 28 .

In sum, these results revealed that the two agents had the therapeuticeffects on the FTLD-involved phenotype through the inhibition of theb-raf pathway.

Example 9

<Analysis on Recovery Effect of b-raf Inhibitor and Tau Knockdown onSpine Phenotype of FTLD Model Mice>

It is known that, in Alzheimer's disease and FTLD-Tau, tauphosphorylation is involved in formation of paired helical filaments(PHF) and aggregation of this protein in the cytoplasm. Moreover, thereis a report on a pathological relation between tau and amyloid β (Aβ) inAlzheimer's disease. From these findings, tau is normally believed to bean effector molecule located downstream of Aβ.

In addition, it has recently been revealed that, in an initialpathological stage when Aβ exhibits toxicity on synapse function, tauplays a different important role in synaptic spines.

Further, it is also reported that the transition of tau to spine due toa Fyn kinase which phosphorylates NMDAR enhances a calcium concentrationand triggers the destruction of spine cytoskeleton.

Hence, based on the above findings, two hypotheses were proposed andexamined. To be more specific, as the first hypothesis, theaforementioned therapeutic effect by suppressing b-raf phosphorylationwas presumably based on the elimination of protein aggregation in thenerve by vemurafenib or thalidomide. Accordingly, analyzed was therecovery effect of vemurafenib or thalidomide on the protein aggregationin the cerebral cortexes of the PGRN-KI mice.

However, although unillustrated, in an immunohistological analysis onthe brains of the PGRN-KI mice, no significant effect of vemurafenib orthalidomide was observed on FUS and p62 inclusion (IB). Moreover,regarding the transition of TDP43 into the cytoplasm also, no largechange was observed in the PGRN-KI mice in which vemurafenib orthalidomide was administered.

Hence, next, the second hypothesis was proposed that synaptic spineswere impaired by abnormal tau phosphorylation. To be more specific, thetherapeutic effect of vemurafenib or thalidomide was presumablyexhibited through the recovery of synaptic spines by suppressing b-rafphosphorylation, and in vivo imaging was performed on synaptic spineswith a two-photon microscope.

Concretely, EGFP-expressing AAV was injected into retrosplenial cortexes(RSD) of the PGRN-KI mice and the C57BL/6J mice. Then, two weeksthereafter, in vivo imaging was performed on synaptic spines with atwo-photon microscope. FIGS. 29 and 30 show the obtained result.

As shown in FIG. 29 , the result of the in vivo imaging on EGFP-positivenerve cells in layer 1 revealed that the spine density was remarkablyreduced in the PGRN-KI mice. Note that the other spine parameters suchas length, diameter, and volume were not much different from those ofthe C57BL/6J mice. These suggest that the reduction in the spine densityis a phenotype requiring a comparatively long time.

Further, as shown in FIG. 30 , the result of observing the spinedynamics at three time points showed that, consistently, the numbers ofproduced spines, eliminated spines, and stably remaining spines did notsignificantly change at any time point within 24 hours.

In addition, analyzed was whether or not vemurafenib and/or tauknockdown enabled recovery of spine related phenotype. FIGS. 31 and 32show the obtained result.

As shown in FIG. 31 , the observation result with a two-photonmicroscope revealed that administering vemurafenib using an osmotic pumprecovered the number of spines of the PGRN-KI mice.

Moreover, as shown in FIG. 32 , the in vivo recovery effect on thenumber of spines was observed as a result of knocking down Tau using alentiviral vector expressing shRNA against tau (sh-tau).

As shown in FIGS. 31 and 32 , the changes in synaptic spines were thesame in the two treatments, but a slight difference was detected in thestatic spine morphology. To be more specific, a trend was observed thatb-raf inhibition decreased the spine volume. On the other hand, the tauknockdown tended to increase the spine volume. This difference betweenthe trends was conceivably because the spines increased by vemurafeniband sh-tau were thin spines in the former, but were thick spines in thelatter. Nevertheless, as shown in FIGS. 31 and 32 , the difference wasnot stably confirmed as a statistically significant difference.

In addition, the spine dynamics were also observed. However, as shown inFIG. 33 , no change was detected in spine production or elimination.

The above results revealed that the vemurafenib administration or tauknockdown had a recovery effect on the number of spines. Moreover, theseresults confirmed that activating the MAPK pathway including tau was amajor mechanism of the abnormal behavior in the PGRN-KI mice.

INDUSTRIAL APPLICABILITY

As has been described above, it has been revealed that, in the pre-onsetstage of Alzheimer's disease, stepwise enhancement of thephosphorylation of the AD core network composed of MARCKS, Marcksl1,SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL,GPRIN1, ACON, ATPA, and ATPB affects dendritic spine dynamics and thelike, consequently developing Alzheimer's disease. Moreover, it has alsobeen revealed that the phosphorylation of the AD core network is causedby PKC, CaMK, CSK, and Lyn, and further that b-raf is involved in thepromotion of the transition from amyloid pathology to tau pathology(enhancement of the phosphorylation of the tau protein) important forthe progression of Alzheimer's disease.

Thus, the present invention targets the proteins composing the AD corenetwork and kinases which phosphorylate these proteins, and is useful inproviding early-stage diagnosis and treatment methods againstAlzheimer's disease and agents utilizable in these methods.

In addition, regarding frontotemporal lobar degeneration (FTLD) also, ithas been revealed that TNF-related signal transduction pathways,particularly a MAPK signal transduction pathway, are activated from apre-onset stage of the disease, and that the activation decreases thenumber of synaptic spines in FTLD patients, consequently developingabnormal behaviors and the like.

Thus, the present invention targets b-RAF belonging to the MAPK signaltransduction pathway, and is useful in providing diagnosis and treatmentmethods against FTLD and agents utilizable in these methods.

1. A method for diagnosing Alzheimer's disease of a test subject, themethod comprising: (i) a step of detecting an activity or expression ofa kinase protein in the test subject; (ii) a step of comparing theactivity or expression with an activity or expression of a kinaseprotein in a normal subject; and (iii) a step of determining that thetest subject is affected with Alzheimer's disease or has a risk ofdeveloping Alzheimer's disease if the activity or expression of thekinase protein in the test subject is higher than the activity orexpression of the kinase protein in the normal subject as a result ofthe comparison, wherein the kinase protein is one or more kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF. 2.An agent for diagnosing Alzheimer's disease, the agent comprising acompound having an activity of binding to one or more kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF. 3.A screening method for a candidate compound for diagnosing Alzheimer'sdisease, the method comprising the steps of: bringing a test compoundinto contact with one or more kinase protein selected from the groupconsisting of PKC, CaMK, CSK, Lyn, and b-RAF; and selecting the compoundif the compound binds to the kinase protein.
 4. An agent for treatingAlzheimer's disease, the agent comprising a compound capable ofsuppressing an activity or expression of one or more kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF. 5.The agent according to claim 4, wherein the compound is capable ofsuppressing an activity or expression of b-RAF and is one or morecompound selected from the group consisting of PLX-4720, sorafenib,GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate, and LGX818. 6.The agent according to claim 5, wherein the compound is vemurafenib. 7.An agent for treating Alzheimer's disease, the agent comprising acompound capable of suppressing a binding of one or more substrateprotein selected from the group consisting of MARCKS, Marcksl1, SRRM2,SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON,ATPA, and ATPB to one or more kinase protein selected from the groupconsisting of PKC, CaMK, CSK, Lyn, and b-RAF.
 8. A screening method fora candidate compound for treating Alzheimer's disease, the methodcomprising: (i) a step of applying a test compound to a system capableof detecting an activity or expression of one or more kinase proteinselected from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF;and (ii) a step of selecting the compound if the compound suppresses theactivity or expression of the protein.
 9. A screening method for acandidate compound for treating Alzheimer's disease, the methodcomprising the following steps (a) to (c): (a) a step of bringing one ormore kinase protein selected from the group consisting of PKC, CaMK,CSK, Lyn, and b-RAF into contact with one or more substrate proteinselected from the group consisting of MARCKS, Marcksl1, SRRM2, SPTA2,ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA,and ATPB, in the presence of a test compound; (b) a step of detecting abinding between the kinase protein and the substrate protein; and (c) astep of selecting the test compound if the test compound suppresses thebinding.